Medical uses of a selective estrogen receptor modulator in combination with sex steroid precursors

ABSTRACT

Novel methods for the medical treatment and/or inhibition of the development of osteoporosis, breast cancer, hypercholesterolemia, hyperlipidemia or atherosclerosis in susceptible warm-blooded animals including humans involving administration of selective estrogen receptor modulator particularly compounds having the general structure: 
                         
and an amount of a sex steroid precursor selected from the group consisting of dehydroepiandrosterone, dehydroepiandrosterone sulfate, androst-5-ene-3β,17β-diol and compounds converted in vivo to one of the foregoing precursor. Further administration of bisphosphonates in combination with selective estrogen receptor modulators and/or sex steroid precursor is disclosed for the medical treatment and/or inhibition of the development of osteoporosis. Pharmaceutical compositions for delivery of active ingredient(s) and kit(s) useful to the invention are also disclosed.

CROSS REFERENCE TO RELATED APPLICATION(S)

This is a divisional of U.S. patent application Ser. No. 10/749,981,filed Dec. 30, 2003, which is a divisional of U.S. patent applicationSer. No. 09/330,799 filed Jun. 11, 1999, now U.S. Pat. No. 6,670,346,issued Dec. 30, 2003, which is a Continuation-in-part application ofU.S. patent application Ser. No. 09/096,284, filed Jun. 11, 1998, nowU.S. Pat. No. 6,465,445 which issued Oct. 15, 2002 in the name ofFernand LABRIE and entitled “MEDICAL USES OF A SELECTIVE ESTROGENRECEPTOR MODULATOR IN COMBINATION WITH SEX STEROID PRECURSORS”.

FIELD OF THE INVENTION

The present invention relates to a method for treating or reducing thelikelihood of acquiring osteoporosis, hypercholesterolemia,hyperlipidemia or atherosclerosis using a novel combination therapy onsusceptible warm-blooded animals, including humans. In particular, thecombination includes administering a selective estrogen receptormodulator (SERM) and raising the patient's level of precursor to sexsteroids, said precursor being selected from the group consisting ofdehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEA-S),and androst-5-ene-3β,17β-diol (5-diol). The invention also relates tokits and pharmaceutical composition for practicing the foregoingcombination.

BACKGROUND OF THE RELATED ART

Man is thus unique, with some other primates, in having adrenals thatsecrete large amounts of the precursor steroids dehydroepiandrosteronesulfate (DHEA-S) and dehydroepiandrosterone (DHEA) which are convertedinto androstenedione (4-dione) and then into active androgens and/orestrogens in peripheral tissues (Labrie et al., In: Important Advancesin Oncology. Edited by V. T. de Vita, S. Hellman, S. A. Rosenberg. J. B.Lippincott, Philadelphia, 193-217, 1985; Labrie, Mol. Cell. Endocrinol.,78: C113-C118, 1991; Labrie, et al., In Signal Transduction inTesticular Cells. Ernst Schering Research Foundation Workshop. Edited byV. Hansson, F. O. Levy, K. Taskén. Springer-Verlag, Berlin-New York(Suppl. 2), pp. 185-218, 1996; Labrie et al., Steroids, 62: 148-158,1997). In a recent study (Labrie, et al., J. Clin. Endocrinol. Metab.,82: 2403-2409, 1997), we have described a dramatic decline in thecirculating levels of dehydroepiandrosterone (DHEA), DHEA-sulfate(DHEA-S), androst-5-ene-3β,17β-diol (5-diol), 5-diol-S, 5-diol fattyacid esters, and androstenedione in both men and women between the agesof 20 and 80 years.

Despite the marked fall in endogenous androgens in women during aging,the use of androgens in post-menopausal women has been limited mainlybecause of the fear of an increased risk of cardiovascular disease asbased upon older studies showing an unfavorable lipid profile withandrogens. Recent studies, however, have shown no significant effect ofcombined estrogen and androgen therapy on the serum levels ofcholesterol, triglycerides, HDL, LDL, and HDL/LDL ratio when compared toestrogen alone (Sherwin et al., Am. J. Obstet. Gynecol., 156: 414-419,1987). In agreement with these observations, we have shown that DHEA, acompound having a predominantly androgenic influence, has apparently nodeleterious effect on the serum lipid profile (Diamond, et al., J.Endocrinol., 150: S43-S50, 1996). Similarly, no change in theconcentrations of cholesterol, its subfractions or triglycerides, over atreatment with estradiol alone has been observed after 6 months oftherapy with estradiol+testosterone implants (Burger et al., Br Med. J.Clin. Res. Ed., 294: 936-937, 1987). It should be mentioned that a studyin man has shown an inverse correlation between serum DHEA-S and lowdensity lipoproteins (Parker et al., Science, 208: 512-514, 1980). Morerecently, a correlation has been found between low serum testosteroneand DHEA and increased visceral fat, a parameter of highercardiovascular risk (Tchernof et al., Metabolism, 44: 513-519, 1995).

Five-diol is a compound biosynthesized from DHEA through the action ofreductive 17β-hydroxysteroid dehydrogenase (17β-HSD) and is a weakestrogen. It has an 85-fold lower affinity than 17β-estradiol (E₂) forthe estrogen receptor in rat anterior pituitary gland cytosol (Simardand Labrie, J. Steroid Biochem., 26: 539-546, 1987), further confirmingthe data obtained on the same parameter in human myometrial and breastcancer tissue (Kreitmann and Bayard, J. Steroid Biochem., 11: 1589-1595,1979; Adams et al., Cancer Res., 41: 4720-4926, 1981; Poulin and Labrie,Cancer Res., 46: 4933-4937, 1986). However, at concentrations wellwithin the range of the plasma levels found in adult women, 5-diolenhances cell proliferation and progesterone receptor levels in humanmammary tumor ZR-75-1 cells (Poulin and Labrie, Cancer Res., 46:4933-4937, 1986) and increases the estrogen-dependent synthesis of the52 kDa glycoprotein in MCF-7 cells (Adams et al., Cancer Res., 41:4720-4926, 1981).

As mentioned above, it is known that the serum levels of DHEA, DHEA-Sand 5-diol decrease with age and correspondingly, that there is adramatic age-dependent reduction in the formation of androgens andestrogens in peripheral target tissues. Such changes in DHEA-S and DHEAsecretion result in a marked decrease in the biochemical and cellularfunctions stimulated by sex steroids. As a result, DHEA and DHEA-S haverecently been used in the treatment of a variety of conditions which areassociated with decrease and/or imbalance in the levels of sex steroids.

Osteoporosis, a condition which affects both men and women, isassociated with a decrease in androgens and estrogens. Estrogens havebeen shown to decrease the rate of bone degradation while androgens havebeen shown to build bone mass. However, estrogen replacement therapycommonly used against osteoporosis requires the addition of progestinsto counteract the endometrial proliferation and the risk of endometrialcancer induced by estrogens. Moreover, since both estrogens andprogestins are thought to increase the risk of breast cancer (Bardon etal., J. Clin. Endocrinol. Metab., 60: 692-697, 1985; Colditz et al., N.Engl. J. Med., 332: 1589-1593, 1995), the use of estrogen-progestinreplacement therapy is accepted by a limited number of women and,usually, for too short periods of time.

Several studies suggest that osteoporosis is a clinical manifestation ofandrogen deficiency in men (Baran et al., Calcif. Tissue Res. 26:103-106, 1978; Odell and Swerdloff, West J. Med. 124: 446-475, 1976;Smith and Walker, Calif. Tissue Res. 22 (Suppl.): 225-228, 1976).Androgen therapy, as observed with nandrolone decanoate, has been foundto increase vertebral bone mineral density in postmenopausal women (Needet al., Arch. Intern. Med., 149: 57-60, 1989). Therapy of postmenopausalwomen with nandrolone increased cortical bone mineral content (Need etal., Clin. Orthop. 225: 273-278, 1987). Androgenic side-effects,however, were recorded in 50% of patients. Such data are of interestsince while almost all present therapies are limited to a reduction ofbone loss, an increase in bone mass was found with the use of theanabolic steroid nandrolone. A similar stimulation of bone formation byandrogens has been suggested in a hypogonadal male (Baran et al.,Calcif. Tissue Res. 26: 103, 1978). A stimulation of bone formation inpostmenopausal women treated with DHEA for 12 months is reported inLabrie et al. (J. Clin. Endocrinol. 82: 3498-3505, 1997).

DHEA (450 mg/kg, b.w., 3 times a week) markedly delayed the appearanceof breast tumors in C3H mice which were genetically bred to developbreast cancer (Schwartz, Cancer Res. 39: 1129-1132, 1979). Moreover, therisk of developing bladder cancer was found to be increased in menhaving lower serum DHEA levels (Gordon et al., Cancer Res. 51:1366-1369, 1991).

U.S. patent application U.S. Pat. No. 5,550,107 relates to a method oftreatment of breast and endometrial cancer in susceptible warm-bloodedanimals which may include inhibition of ovarian hormonal secretion bysurgical means (ovariectomy) or chemical means (use of an LHRH agonist,e.g. [D-Trp⁶, des-Gly-NH₂ ¹⁰]LHRH ethylamide, or antagonist) as part ofa combination therapy. Antiestrogens, androgens, progestins, inhibitorsof sex steroid formation (especially of 17β-hydroxysteroiddehydrogenase- or aromatase-catalyzed production of sex steroids),inhibitors of prolactin secretion and of growth hormone secretion andACTH secretion are discussed. A counterpart thereof has been publishedunder international publication number WO 90/10462.

In addition, cardiovascular diseases have been associated with decreasedserum levels of DHEA and DHEA-S and both DHEA and DHEA-S have beensuggested to prevent or treat these conditions (Barrett-Connor et al.,N. Engl. J. Med. 315: 1519-1524, 1986).

In aged Sprague-Dawley rats, Schwartz (in Kent, Geriatrics 37: 157-160,1982) has observed that body weight was reduced from 600 to 550 g byDHEA without affecting food intake. Schwartz (Cancer 39: 1129-1132,1979) observed that C3H mice given DHEA (450 mg/kg, 3 times a week)gained significantly less weight and grew older than the controlanimals, had less body fat and were more active. The reduction in bodyweight was achieved without loss of appetite or food restriction.Furthermore, DHEA could prevent weight gain in animals bred to becomeobese in adulthood (in Kent, Geriatrics 37: 157-160, 1982).

DHEA administration to lean Zucher rats decreased body weight gaindespite increased food intake. Treated animals had smaller fat padsthus, overall, suggesting that DHEA increases food metabolism, resultingin lower weight gain and fat accumulation (Svec et al., Proc. 2^(nd)Int. Conf. Cortisol and Anti-Cortisols, Las Vegas, Nev., USA, p. 56abst., 1997).

Obesity was found to be improved in the A^(vy) mutant mouse (Yen et al.,Lipids 12: 409-413, 1977) and in the Zucker rat (Cleary and Zisk, Fed.Proc. 42: 536, 1983). DHEA-treated C3H mice had a younger appearancethan controls (Schwartz, Cancer Res. 39: 1129-1132, 1979).

DHEA reduced the incidence of atherosclerosis in cholesterol-fed rabbits(Gordon et al., J. Clin. Invest. 82: 712-720, 1988; Arad et al.,Arteriosclerosis 9: 159-166, 1989). Moreover, high serum concentrationsof DHEA-S have been reported to protect against death fromcardiovascular diseases in men (Barrett-Connor et al., N. Engl. J. Med.315: 1519-1524, 1986). Circulating levels of DHEA and DHEA-S have thusbeen found to be inversely correlated with mortality from cardiovasculardisease (Barrett-Connor et al., N. Engl. J. Med. 315: 1519-1524, 1986)and to decrease in parallel with the diminished immune competence(Thoman and Weigle, Adv. Immunol. 46: 221-222, 1989). A study in man hasshown an inverse correlation between fetal serum DHEA-S and low densitylipoprotein (LDL) levels (Parker et al., Science 208: 512, 1980).

Uses of DHEA as well as the benefits of androgen and estrogen therapyare discussed in International Patent Publication WO 94/16709.

Correlations observed in the prior art are not believed to suggesttreatment or prophylactic methods that are effective, or as free ofundesirable side-effects, as are combination therapies disclosed here.

SUMMARY OF THE INVENTION

It is accordingly an object of the present invention to provideeffective methods of treatment for osteoporosis, hypercholesterolemia,hyperlipidemia, atherosclerosis, breast cancer, endometrial cancer,ovarian cancer and uterine cancer while minimizing undesirable sideeffects.

It is another object to provide methods of reducing the risk ofacquiring the above diseases.

It is another object to provide kits and pharmaceutical compositionssuitable for use in the above methods.

In one embodiment, the invention pertains to a method of treating orreducing the risk of acquiring osteoporosis comprising increasing levelsof a sex steroid precursor selected from the group consisting ofdehydroepiandrosterone (DHEA), dehydroepiandrosterone-sulfate (DHEA-S)and androst-5-ene-3β,17β-diol (5-diol), in a patient in need of saidtreatment or said reduction, and further comprising administering tosaid patient a therapeutically effective amount of a selective estrogenreceptor modulator (SERM) as part of a combination therapy.

In another embodiment, the invention provides a method of treating orreducing the risk of acquiring hypercholesterolemia comprisingincreasing levels of a sex steroid precursor selected from the groupconsisting of dehydroepiandrosterone, dehydroepiandrosterone-sulfate andandrost-5-ene-3β,17β-diol, in a patient in need of said treatment orsaid reduction, and further comprising administering to said patient atherapeutically effective amount of a selective estrogen receptormodulator as part of a combination therapy.

In another embodiment, the invention provides a method of treating orreducing the risk of acquiring hyperlipidemia comprising increasinglevels of a sex steroid precursor selected from the group consisting ofdehydroepiandrosterone, dehydroepiandrosterone-sulfate andandrost-5-ene-3β,17β-diol, in a patient in need of said treatment orsaid reduction, and further comprising administering to said patient atherapeutically effective amount of a selective estrogen receptormodulator as part of a combination therapy.

In another embodiment, the invention provides a method of treating orreducing the risk of acquiring atherosclerosis comprising increasinglevels of a sex steroid precursor selected from the group consisting ofdehydroepiandrosterone, dehydroepiandrosterone-sulfate andandrost-5-ene-3β,17β-diol, in a patient in need of said treatment orsaid reduction, and further comprising administering to said patient atherapeutically effective amount of a selective estrogen receptormodulator as part of a combination therapy.

In another embodiment, the invention provides a method of treating orreducing the risk of acquiring breast cancer comprising increasinglevels of a sex steroid precursor selected from the group consisting ofdehydroepiandrosterone, dehydroepiandrosterone-sulfate andandrost-5-ene-3β,17β-diol, in a patient in need of said treatment orsaid reduction, and further comprising administering to said patient atherapeutically effective amount of a selective estrogen receptormodulator as part of a combination therapy.

In another embodiment, the invention provides a method of treating orreducing the risk of acquiring endometrial cancer comprising increasinglevels of a sex steroid precursor selected from the group consisting ofdehydroepiandrosterone, dehydroepiandrosterone-sulfate andandrost-5-ene-3β,17β-diol, in a patient in need of said treatment orsaid reduction, and further comprising administering to said patient atherapeutically effective amount of a selective estrogen receptormodulator as part of a combination therapy.

In another embodiment, the invention provides a method of treating orreducing the risk of acquiring uterine cancer comprising increasinglevels of a sex steroid precursor selected from the group consisting ofdehydroepiandrosterone, dehydroepiandrosterone-sulfate andandrost-5-ene-3β,17β-diol, in a patient in need of said treatment orsaid reduction, and further comprising administering to said patient atherapeutically effective amount of a selective estrogen receptormodulator as part of a combination therapy.

In another embodiment, the invention provides a method of treating orreducing the risk of acquiring ovarian cancer comprising increasinglevels of a sex steroid precursor selected from the group consisting ofdehydroepiandrosterone, dehydroepiandrosterone-sulfate andandrost-5-ene-3β,17β-diol, in a patient in need of said treatment orsaid reduction, and further comprising administering to said patient atherapeutically effective amount of a selective estrogen receptormodulator as part of a combination therapy.

In another embodiment, the invention provides a kit comprising a firstcontainer containing a therapeutically effective amount of at least onesex steroid precursor selected from the group consisting ofdehydroepiandrosterone, dehydroepiandrosterone-sulfate,androst-5-ene-3β,17β-diol and any prodrug that is converted in vivo intoany of the foregoing precursors; and further comprising a secondcontainer containing a therapeutically effective amount of at least oneselective estrogen receptor modulator.

In another embodiment, the invention provides a pharmaceuticalcomposition comprising: a) a pharmaceutically acceptable excipient,diluent or carrier; b) a therapeutically effective amount of at leastone sex steroid precursor selected from the group consisting ofdehydroepiandrosterone, dehydroepiandrosterone-sulfate,androst-5-ene-3β,17β-diol and a prodrug that is converted in vivo intoany of the foregoing sex steroid precursors; and c) a therapeuticallyeffective amount of at least one selective estrogen receptor modulator.

In another embodiment, the invention provide a method of reducing therisk of acquiring breast cancer comprising administering, to a patientin need of such reduction a prophylactically effective amount of aselective estrogen receptor modulator.

In one embodiment of reducing the likelihood of acquiring breast cancer,it is desirable to combine administration of a SERM with administrationof a sex steroid precursor. However, the invention also includesadministering a SERM alone, which is shown, for example, in FIGS. 1 and2, to provide significant prophylactic effect, even in the absence ofadministered precursors. Preferred SERMs for this purpose are the sameas discussed for other uses herein. Preferred dosages and methods ofadministration are also the same.

As used herein, a selective estrogen receptor modulator (SERM) is acompound that either directly or through its active metabolite functionsas an estrogen receptor antagonist (“antiestrogen”) in breast tissue,yet provides estrogenic or estrogen-like effect on bone tissue and onserum cholesterol levels (i.e. by reducing serum cholesterol).Non-steroidal compounds that function as estrogen receptor antagonistsin vitro or in human or rat breast tissue (especially if the compoundacts as an antiestrogen on human breast cancer cells) is likely tofunction as a SERM. Conversely, steroidal antiestrogens tend not tofunction as SERMs because they tend not to display any beneficial effecton serum cholesterol. Non-steroidal antiestrogens we have tested andfound to function as SERMs include EM-800, EM-01538, Raloxifene,Tamoxifen and Droloxifene. We have tested the steroidal antiestrogen ICI182,780 and found not to function as SERMs. SERMs in accordance with theinvention may be administered in the same dosage as known in the artwhen these compounds are used as antiestrogens.

We have also noted a correlation between the beneficial effect SERMshave on serum cholesterol and beneficial estrogenic or estrogen-likeeffects on bone and on serum lipids. SERMs that have been shown in ourresearch to act beneficially on all of these parameters, include bonemass, cholesterol, and triglyceride levels. Without intending to bebound by theory, it is believed that SERMs, many of which preferably,have two aromatic rings linked by one to two carbon atoms, are expectedto interact with the estrogen receptor by virtue of the foregoingportion of the molecule that is best recognized by the receptor.Preferred SERMs have side chains which may selectively causeantagonistic properties in breast tissue without having significantantagonistic properties in other tissues. Thus, the SERMs may desirablyfunctions as antiestrogens in the breast while surprisingly anddesirably functioning as estrogens (or providing estrogen-like activity)in bone and in the blood (where concentrations of lipid and cholesterolare favorably affected). The favorable effect on cholesterol and lipidtranslates to a favorable effect against atherosclerosis which is knownto be adversely, affected by improper levels of cholesterol and lipid.

All of the diseases treated by the invention as discussed herein respondfavorably to androgens. Rather than utilizing androgens per se,applicants utilize sex steroid precursors such as DHEA, DHEA-S, 5-diol,or prodrugs converted to any such sex steroid precursors. In vivo,DHEA-S is converted to DHEA which in turn converts to 5-diol. It isbelieved that any tissue responding favorably to one is likely torespond favorably to the others. Prodrug forms of active metabolites arewell known in the art. See, e.g. H. Bundgaard “Design and Application ofProdrugs” (In: A Textbook of Drug Design and Development. Edited by H.Bundgaard and P. Krogsgaard-Larsen; Harwook Academic Publishers GmfH,Chur: Switzerland, 1991), the contents of which are incorporated hereinby reference. In particular, see pages 154-155 describing variousfunctional groups of active metabolites and appropriate correspondingprodrug groups that convert in vivo to each functional group. Where apatients' levels of sex steroid precursors are raised in accordance withthe invention, that may typically be accomplished by administering sucha precursor or by administering a prodrug of such a precursor. Byutilizing precursors instead of androgens, undesirable androgenicactivity in tissues other than the target is reduced. Tissues convertprecursors such as DHEA to androgens only through a natural and moreregulated process. A large percentage of androgens are locally producedin peripheral tissues and to different extents in different tissues.

The cancers treated in accordance with the invention respond adverselyto estrogenic activity. On the other hand, osteoporosis,hypercholesterolemia, hyperlipidemia, and atherosclerosis respondfavorably to estrogenic or estrogen-like activity. By using SERMs inaccordance with the invention, desirable effects are provided in targettissues without undesirable effects in certain other tissues. Forexample, a SERM can have favorable estrogenic effect in the bone (or onlipid or cholesterol) while avoiding unfavorable estrogenic effect inthe breast.

Thus both precursor and SERM provide favorable effect in target tissueswhile minimizing unfavorable effects in certain other tissues. Moreover,there are substantial synergies in using the two together in accordancewith the invention. For example, estrogens and androgens providebeneficial effect against osteoporosis by different mechanisms (estrogenreducing bone resorption, androgen contributing to bone formation). Thecombination of the present invention provides bone with beneficialestrogen or estrogen-like effect through the activity of SERM, and alsoprovides beneficial androgen through local conversion of precursor toandrogen in the bone. Precursor is also believed to provide estrogen.The same is true in connection with controlling lipid or cholesterol(useful for treating or preventing atherosclerosis). A similar synergyis provided against breast, endometrial, ovarian or uterine cancer wherethe SERM provides desirable antiestrogenic effect and the precursorprovides desirable androgenic effect (with any incidental conversion ofprecursor to estrogen being mitigated by the antiestrogen). Undesirableeffects are also mitigated in a synergistic way by the combination usedin the invention. For all diseases discussed herein, any other effect onbreast tissues that might otherwise result from estrogens produced bythe precursor (when the precursor is used for promoting androgeniceffects in accordance with the invention) is mitigated by theantiestrogenic effect of the SERM in breast tissue.

In some embodiments, progestins are added to provide further androgeniceffect. Progestins may be used at low dosages known in the art withoutadversely affecting receptors other than the androgen receptors (e.g.glucocorticoid receptors). They also are relatively free of unwantedandrogenic side effects (such as facial hair with female patients).

Preferred SERMs discussed herein relate: (1) to all diseases stated tobe susceptible to the invention; (2) to both therapeutic andprophylactic applications; and (3) to preferred pharmaceuticalcompositions and kits.

In one embodiment, the precursor is DHEA.

In another embodiment, the precursor is DHEA-S.

In another embodiment, the precursor is 5-diol.

A patient in need of treatment or of reducing the risk of onset of agiven disease is one who has either been diagnosed with such disease orone who is susceptible to acquiring such disease.

Except where otherwise stated, the preferred dosage of the activecompounds (concentrations and modes of administration) of the inventionis identical for both therapeutic and prophylactic purposes. The dosagefor each active component discussed herein is the same regardless of thedisease being treated (or of the disease whose likelihood of onset isbeing reduced).

Except when otherwise noted or where apparent from context, dosagesherein refer to weight of active compounds unaffected by pharmaceuticalexcipients, diluents, carriers or other ingredients, although suchadditional ingredients are desirably included, as shown in the examplesherein. Any dosage form (capsule, tablet, injection or the like)commonly used in the pharmaceutical industry is appropriate for useherein, and the terms “excipient”, “diluent”, or “carrier” include suchnonactive ingredients as are typically included, together with activeingredients in such dosage forms in the industry. For example, typicalcapsules, pills, enteric coatings, solid or liquid diluents orexcipients, flavorants, preservatives, or the like may be included.

All of the active ingredients used in any of the therapies discussedherein may be formulated in pharmaceutical compositions which alsoinclude one or more of the other active ingredients. Alternatively, theymay each be administered separately but sufficiently simultaneous intime so that a patient eventually has elevated blood levels or otherwiseenjoys the benefits of each of the active ingredients (or strategies)simultaneously. In some preferred embodiments of the invention, forexample, one or more active ingredients are to be formulated in a singlepharmaceutical composition. In other embodiments of the invention, a kitis provided which includes at least tow separate containers wherein thecontents of at least one container differs, in whole or in part, fromthe contents of at least one other container with respect to activeingredients contained therein.

Combination therapies discussed herein also include use of one activeingredient (of the combination) in the manufacture of a medicament forthe treatment (or risk reduction) of the disease in question where thetreatment or prevention further includes another active ingredient ofthe combination in accordance with the invention. For example in oneembodiment, the invention provides the use of a SERM in the preparationof a medicament for use, in combination with a sex steroid precursorselected from the group consisting of DHEA, DHEA-S, 5-diol, andpro-drugs converted to any of the foregoing sex steroid precursors, invivo, in the treatment of any of the diseases for which the presentcombination therapy is believed effective (i.e., breast cancer,endometrial cancer, uterine cancer, ovarian cancer, osteoporosis,hypercholesterolemia, hyperlipidemia, and atherosclerosis). In anotherembodiment, the invention provides the use of a sex steroid precursorselected from the group consisting of DHEA, DHEA-S, 5-diol, andpro-drugs converted to any of the foregoing sex steroid precursors, invivo, in the preparation of a medicament for use, in combination with aSERM, for treatment of any of those same diseases.

In one embodiment of the invention, DHEA is not utilized as theprecursor. In another embodiment, EM-800 is not used as the SERM. Inanother embodiment, the combination of DHEA with EM-800 is not used.

In one preferred embodiment, DHEA is used in combination with EM-1538.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of treatment with DHEA (10 mg, percutaneously,once daily) or EM-800 (75 μg, orally, once daily) alone or incombination for 9 months on the incidence of DMBA-induced mammarycarcinoma in the rat throughout the 279-day observation period. Data areexpressed as percentage of the total number of animals each group.

FIG. 2 shows the effect of treatment with DHEA (10 mg, percutaneously,once daily) or EM-800 (75 μg, orally, once daily) alone or incombination for 9 months on average tumor number per tumor-bearinganimal (A) and on average tumor size per tumor-bearing rat (B)throughout the 279-day observation period. Data are expressed as themeans±SEM.

FIG. 3 shows the effect of treatment with DHEA (10 mg, percutaneously,once daily) or EM-800 (75 μg, orally, once daily) alone or incombination for 9 months on serum triglyceride (A) and cholesterol (B)levels in the rat. Data are expressed as the means±SEM. **: P<0.01experimental versus respective control.

FIG. 4 shows: A) Effect of increasing doses of DHEA (0.3 mg, 1.0 mg or3.0 mg) administered percutaneously twice daily on average ZR-75-1 tumorsize in ovariectomized (OVX) nude mice supplemented with estrone.Control OVX mice receiving the vehicle alone are used as additionalcontrols. The initial tumor size was taken as 100%. DHEA wasadministered percutaneously (p.c.) in a 0.02 ml solution of 50%ethanol-50% propylene glycol on the dorsal skin. B) Effect of treatmentwith increasing doses of DHEA or EM-800 alone or in combination for 9.5months on ZR-75-1 tumor weight in OVX nude mice supplemented withestrone. **, p<0.01, treated versus control OVX mice supplemented withestrone.

FIG. 5 shows the effect of increasing oral doses of the antiestrogenEM-800 (15 μg, 50 μg or 100 μg) (A) or of percutaneous administration ofincreasing doses of DHEA (0.3, 1.0 or 3.0 mg) combined with EM-800 (15μg) or EM-800 alone (B) for 9.5 months on average ZR-75-1 tumor size inovariectomized (OVX) nude mice supplemented with estrone. The initialtumor size was taken as 100%. Control OVX mice receiving the vehiclealone were used as additional controls. Estrone was administeredsubcutaneously at the dose of 0.5 μg once daily while DHEA was dissolvedin 50% ethanol-50% propylene glycol and applied on the dorsal skin areatwice daily in a volume of 0.02 ml. Comparison is also made with OVXanimals receiving the vehicle alone.

FIG. 6 shows the effect of 12-month treatment withdehydroepiandrosterone (DHEA) alone or in combination with Flutamide orEM-800 on trabecular bone volume in ovariectomized rats. Intact animalsare added as additional controls. Data are presented as mean±SEM**p<0.01 versus OVX Control.

FIG. 7 shows the effect of 12-month treatment withdehydroepiandrosterone (DHEA) alone or in combination with Flutamide orEM-800 on trabecular number in ovariectomized rats. Intact animals areadded as additional controls. Data are presented as mean±SEM **p<0.01versus OVX Control.

FIG. 8 shows proximal tibia metaphyses from intact control (A),ovariectomized control (B), and ovariectomized rats treated with DHEAalone (C) or in combination with Flutamide (D) or EM-800 (E). Note thereduced amount of trabecular bone (T) in ovariectomized control animals(B), and the significant increase in trabecular bone volume (T) inducedafter DHEA administration (C). The addition of Flutamide to DHEApartially blocked the effect of DHEA on the trabecular bone volume (D),whereas the combination of DHEA and EM-800 provided complete protectionagainst the ovariectomy-associated bone loss. Modified trichromeMasson-Goldner, magn.×80. T: Trabeculae, GP: Growth Plate.

FIG. 9 shows the effect of increasing doses (0.01, 0.03, 0.1, 0.3, and 1mg/kg) of EM-800, EM-1538 and Raloxifene (EM-1105) administered per osdaily for 4 days on cholesterol level of ovariectomized rat.

FIG. 10 shows the effect of 34-week treatment withdehydroepiandrosterone (DHEA) alone or in combination with EM-1538(EM-652.HCl) on lumbar spine BMD in ovariectomized rats. Intact animalsare added as additional controls. Data are presented as mean±SEM**p<0.01 versus OVX Control.

FIG. 11 shows the combined effects of the SERM (EM-652) and DHEA onparameters of menopause. No negative effect is expected.

FIG. 12 shows the plasma concentration of DHEA (ng/mL) (Y axis) infunction of time (X-axis) after a single oral absorption of preferredsex steroid precursors of the invention (150 μmol/rat) in male rats. Inthe box, AUC 24 h of DHEA induced by these compounds is reported.

EM-760 dehydroepiandrosterone

EM-900 androst-5-ene-3β,17β-diol

EM-1304 androst-5-ene-3β,17β-diol 3-acetate

EM-1305-CS androst-5-ene-3β,17β-diol diacetate

EM-1397 androst-5-ene-3β,17β-diol 3 acetate 17 benzoate

EM-1400 androst-5-ene-3β,17β-diol dibenzoate

EM-1410 androst-5-ene-3β,17β-diol dipropionate

EM-1474-D androst-5-ene-3β,17β-diol dihemisuccinate

FIG. 13 shows the plasma concentration of androst-5-ene-3β,17β-diol(ng/mL) (Y axis) in function of time (X-axis) after a single oralabsorption of sex steroid precursor of the invention (150 μmol/rat) inmale rats. In the box, AUC 24 h of androst-5-ene-3β,17β-diol induced bythese compounds is reported.

EM-760 dehydroepiandrosterone

EM-900 androst-5-ene-3β,17β-diol

EM-1304 androst-5-ene-3β,17β-diol 3-acetate

EM-1305-CS androst-5-ene-3β,17β-diol diacetate

EM-1397 androst-5-ene-3β,17β-diol 3 acetate 17 benzoate

EM-1400 androst-5-ene-3β,17β-diol dibenzoate

EM-1410 androst-5-ene-3β,17β-diol dipropionate

EM-1474-D androst-5-ene-3β,17β-diol dihemisuccinate

DETAIL DESCRIPTION OF THE INVENTION

Estrogens are well-known to stimulate the proliferation of breastepithelial cells and cell proliferation itself is thought to increasethe risk of cancer by accumulating random genetic errors that may resultin neoplasia (Preston Martin et al., Cancer. Res. 50: 7415-21, 1990).Based on this concept, antiestrogens have been introduced to preventbreast cancer with the objective of reducing the rate of cell divisionstimulated by estrogens.

The loss of ovarian cyclicity found in female Sprague-Dawley rats after10 months of age is accompanied by increased serum estrogen andprolactin levels and decreased serum androgen and progesteroneconcentrations (Lu et al., 61st Annual Meeting of the Endocrine Society106 (abst. #134), 1979; Tang et al., Biol. Reprod. 31: 399-413, 1984;Russo et al., Monographs on Pathology of Laboratory Animals: Integumentand Mammary Glands 252-266, 1989; Sortino and Wise, Endocrinology 124:90-96, 1989; Cardy, Vet. Pathol. 28: 139-145, 1991). These hormonalchanges that spontaneously occur in aging female rats are associatedwith multifocal proliferation and increased secretory activity of theacinar/alveolar tissue as well as mammary gland duct dilatation andformation of cysts (Boorman et al., 433, 1990; Cardy, Vet. Pathol. 28:139-145, 1991). It should be mentioned that hyperplastic and neoplasticchanges of the rat mammary gland are often accompanied by increasedlevels of estrogens and prolactin (Meites, J. Neural. Transm. 48: 25-42,1980). Treatment with EM-800, a SERM of the present invention, inducesatrophy of the mammary gland which is characterized by a decrease in thesize and number of the lobular structures, and no evidence of secretoryactivity, indicating the potent antiestrogenic activity of EM-800 in themammary gland (Luo et al. Endocrinology 138: 4435-4444, 1997).

Treatment with DHEA, a sex steroid precursor of the present invention,leads to an elevation in serum DHEA and 5-diol while serum 4-dione,testosterone, dihydrotestosterone, and estradiol levels are onlymoderately increased or more often remain unchanged, thus confirming theintracellular biotransformation of this precursor steroid in peripheraltissues (Labrie et al., Mol. Cell. Endocrinol. 78: C113-C118, 1991).However, the stimulatory effect of orally administered DHEA on serumandrogens, such as testosterone and dihydrotestosterone, is of greateramplitude than the effect on serum estrogens, thus suggesting that DHEAis predominantly transformed into androgens in these animals. Thisobservation is in agreement with the data obtained in women where theformation of androgens from DHEA was a more important pathway than theconversion of DHEA into estrogens (Morales et al., J. Clin. Endocrinol.Metab. 78: 1360-1367, 1994; Labrie et al., Ann. N. Y. Acad. Sci. 774:16-28, 1995; Labrie et al., Steroids 62: 148-158, 1997).

With the knowledge of the above-described potent antiestrogenic activityresulting in mammary gland atrophy and the predominant androgenic effectof DHEA on the mammary gland, the histomorphological changes seen inanimals treated with the combination of a SERM and a sex steroidprecursor are best explained by an unopposed androgenic action of DHEAin the rat mammary gland.

Most importantly, it has been observed that androgens exert a directantiproliferative activity on the growth of ZR-75-1 human breast cancercells in vitro and that such an inhibitory effect of androgens isadditive to that of an antiestrogen (Poulin and Labrie, Cancer Res. 46:4933-4937, 1986; Poulin et al., Breast Cancer Res. Treat. 12: 213-225,1988). Similar inhibitory effects have been observed in vivo on ZR-75-1xenographts in nude mice (Dauvois et al., Cancer Res. 51: 3131-3135,1991). Androgens have also been shown to inhibit the growth ofDMBA-induced mammary carcinoma in the rat, this inhibition beingreversed by the simultaneous administration of the pure antiandrogenFlutamide (Dauvois et al., Breast Cancer Res. Treat. 14: 299-306, 1989).Taken together, the present data indicate the involvement of theandrogen receptor in the inhibitory action of DHEA on breast cancer.

Since antiestrogens and sex steroid precursors exert inhibitory effectson breast cancer via different mechanisms, the present invention showsthat the combination of a SERM (EM-800) and a sex steroid precursor(DHEA) exerts more potent inhibitory effects than each compound usedalone on the development of DMBA-induced rat mammary carcinoma as wellillustrated in FIGS. 1 and 2. In fact, no DMBA-induced tumor was foundat the end of the experiment in animals that had received both DHEA andEM-800.

The present invention describes that the combination of a sex steroidprecursor (DHEA) and a SERM (EM-800) maintained the stimulatory effectof DHEA on bone formation and potentiated the inhibitory effect of theSERM (EM-800) alone on bone turnover and resorption as demonstrated bythe further decreases in urinary hydroxyproline and calcium excretionwhen both compounds were combined.

We have shown that DHEA has beneficial effects on bone in both thefemale rat (Luo et al., Endocrinology 138: 4435-4444, 1997), andpostmenopausal women (Labrie et al., J. Clin. Endocrinol. Metab. 82:3498-3505, 1997). Thus, in intact female rats, treatment with DHEAincreases bone mineral density (BMD) of total skeleton, lumbar spine andfemur (Luo et al., Endocrinology 138: 4435-4444, 1997).

On the other hand, treatment with EM-800 had no significant effect onBMD in intact animals although potent stimulatory effects are observedin the ovariectomized rat (Martel et al., unpublished data). SinceEM-800 exerts such stimulatory effects on BMD of total skeleton, lumbarspine and femur in ovariectomized rats, the lack of significantstimulatory effect of EM-800 in intact animals could be due to the factthat the sex steroids present in intact female rats exert maximal effecton BMD (Luo et al., Endocrinology 138: 4435-4444, 1997). Similarly, thelack of significant effect of EM-800 in ovariectomized rats alreadyreceiving DHEA is likely due to the maximal stimulatory effects exertedby the androgens (and possibly estrogens) synthesized in bone cells fromexogenous DHEA.

Estrogens are known to lower serum cholesterol but to increase or tohave no effect on serum triglycerides levels (Love et al., Ann. Intern.Med. 115: 860-864, 1991; Walsh et al., New Engl. J. Med. 325: 1196-1204,1991; Barrett-Connor, Am. J. Med. 95 (Suppl. 5A): 40S-43S, 1993; Russellet al., Atherosclerosis 100: 113-122, 1993; Black et al., J. Clin.Invest. 93: 63-69, 1994; Dipippo et al., Endocrinology 136: 1020-1033,1995; Ke et al., Endocrinology 136: 2435-2441, 1995). FIG. 3 shows thatEM-800 possesses both hypocholesterolemic and hypotriglyceridemiceffects in the rat, thus showing its unique action on the serum lipidprofile which is apparently different from other SERMs, such astamoxifen (Bruning et al., Br. J. Cancer 58: 497-499, 1988; Love et al.,J. Natl. Cancer Inst. 82: 1327-1332, 1990; Dipippo et al., Endocrinology136: 1020-1033, 1995; Ke et al., Endocrinology 136: 2435-2441, 1995),droloxifene (Ke et al., Endocrinology 136: 2435-2441, 1995), andraloxifene (Black et al., J. Clin. Invest. 93: 63-69, 1994). Thecombination of DHEA and EM-800 preserved the hypocholesterolemic andhypotriglyceridemic effects of EM-800, thus suggesting that such acombination could exert beneficial effects on serum lipids.

It should be mentioned that the serum lipid profile is markedlydifferent between rats and humans. However, since an estrogenreceptor-mediated mechanism is involved in the hypocholesterolemiceffect of estrogens as well as antiestrogens (Lundeen et al.,Endocrinology 138: 1552-1558, 1997), the rat remains a useful model tostudy the cholesterol-lowering effect of estrogens and “antiestrogens”in humans.

In brief, the above-described data clearly demonstrate the effects ofthe combination of a SERM (EM-800) and a sex steroid precursor (DHEA) onthe development of mammary carcinoma induced by DMBA as well as theprotective effects of such a combination on bone mass and serum lipids.Such data suggest the additional beneficial effects of such acombination for treatment and prevention of osteoporosis while improvingthe lipid profile.

We have also studied the potential interaction of the inhibitory effectof the novel antiestrogen (EM-800) with that of sex steroid precursor(DHEA) on the growth of human ZR-75-1 breast cancer xenografts in nudemice by combined administration of the two drugs. FIGS. 4 and 5 showthat DHEA, by itself, at the doses used, causes a 50 to 80% inhibitionof tumor growth while the near complete inhibition of tumor growthachieved with a low dose of the antiestrogen was not affected by DHEA.

The limitations of bone mineral density (BMD) measurements are wellknown. As an example, BMD measurements showed no change in rats treatedwith the steroidal antiestrogen ICI 182780 (Wakeling, Breast Cancer Res.Treat. 25: 1-9, 1993) while inhibitory changes were seen byhistomorphometry (Gallagher et al., Endocrinology 133: 2787-2791, 1993).Similar differences were reported with Tamoxifen (Jordan et al., BreastCancer Res. Treat. 10: 31-35, 1987; Sibonga et al., Breast Cancer Res.Treatm. 41: 71-79, 1996).

It should be indicated that reduced bone mineral density is not the onlyabnormality associated with reduced bone strength. (Guidelines forpreclinical and clinical evaluation of agents used in the prevention ortreatment of postmenopausal osteoporosis, Division of Metabolism andEndocrine Drug Products, FDA, May 1994). It is thus important to analyzethe changes in biochemical parameters of bone metabolism induced byvarious compounds and treatments in order to gain a better knowledge oftheir action.

It is particularly important to indicate that the combination of DHEAand EM-800 exerted unexpected beneficial effects on importantbiochemical parameters of bone metabolism. In fact, DHEA alone did notaffect the urinary hydroxyproline/creatinine ratio, a marker of boneresorption. Moreover, no effect of DHEA could be detected on dailyurinary calcium or phosphorus excretion (Luo et al., Endocrinology 138:4435-4444, 1997). EM-800, on the other hand, decreased the urinaryhydroxyproline/creatinine ratio by 48% while, similarly to DHEA, noeffect of EM-800 was seen on urinary calcium or phosphorus excretion.EM-800, moreover, had no effect on serum alkaline phosphatase activity,a marker of bone formation while DHEA increased the value of theparameter by about 75% (Luo et al., Endocrinology 138: 4435-4444, 1997).

One of the unexpected effects of the combination of DHEA and EM-800relates to the urinary hydroxyproline/creatinine ratio, a marker of boneresorption, which was reduced by 69% when both DHEA and EM-800 werecombined, this value being statistically different (p<0.01) from the 48%inhibition achieved by EM-800 alone while DHEA alone did not show anyeffect. Thus, the addition of DHEA to EM-800 increases by 50% of theinhibitory effect of EM-800 on bone reabsorption. Most importantly,another unexpected effect of the addition of DHEA to EM-800 was theapproximately 84% decrease in urinary calcium (from 23.17±1.55 to3.71±0.75 μmol/24 h/100 g (p<0.01) and the 55% decrease in urinaryphosphorus (from 132.72±6.08 to 59.06±4.76 μmol/24 h/100 g (p<0.01)respectively, (Luo et al., Endocrinology 138: 4435-4444, 1997).

TABLE 1 URINE CALCIUM PHOSPHORUS HP/Cr SERUM tALP GROUP (μmol/24 h/100g) (μmol/24 h/100 g) (μmol/mmol) (IU/L) CONTROL 23.17 ± 1.55 132.72 ±6.08 13.04 ± 2.19 114.25 ± 14.04 DHEA (10 mg) 25.87 ± 3.54 151.41 ±14.57 14.02 ± 1.59 198.38 ± 30.76* EM-800 (75 μg) 17.44 ± 4.5 102.03 ±25.13  6.81 ± 0.84** 114.11 ± 11.26 DHEA + EM-800  3.71 ± 0.75**  59.06± 4.76**  4.06 ± 0.28** 204.38 ± 14.20**

It is also of interest to note that the potent inhibitory effect ofEM-800 on serum cholesterol is not prevented by simultaneous treatmentwith DHEA (Luo et al., Endocrinology 138: 4435-4444, 1997).

While Raloxifene and similar compounds prevent bone loss and decreaseserum cholesterol (like estrogens), it should be mentioned that whenRaloxifene was compared to Premarin on BMD, the effect of Raloxifene onBMD was less potent than that of Premarin (Minutes of the Endocrinologyand Metabolism Drugs Advisory Committee, FDA Thursday, Meeting #68, Nov.20, 1997). Because of its well known adverse effects on breast anduterine cancer, the addition of an estrogen to Raloxifene, EM-800 orother similar compounds is not an acceptable solution.

The present results obtained in the rat clearly demonstrate that DHEAcan provide the beneficial effects which are lacking with the use of aselective estrogen receptor modulator (SERM) alone such as EM-800,Raloxifene, etc. While a SERM has effects limited to inhibition of boneresorption, the addition of DHEA, 5-diol, DHEA-S is believed tostimulate bone formation (an effect not found with a SERM or anestrogen) and further reduce bone resorption above the effect achievedwith EM-800.

Importantly, the combination of EM-800 and DHEA in ovariectomized ratstreated for 12 months has beneficial effects on bone morphometry.Trabecular bone volume is particularly important for bone strength andto prevent bone fractures. Thus, in the above-mentioned study,trabecular bone volume of the tibia increased from 4.1±0.7% inovariectomized rats to 11.9±0.06% (p<0.01) with DHEA alone while theaddition of EM-800 to DHEA further increased trabecular bone volume to14.7±1.4%, a value similar to that found in intact controls (FIG. 6)

From a value of 0.57±0.08 per mm in ovariectomized rats, treatment withDHEA resulted in a 137% increase in trabecular bone number compared toovariectomized controls. The stimulatory effect of DHEA thus reached1.27±0.1 per mm while simultaneous treatment with EM-800 and DHEAresulted in an additional 28% increase in trabecular bone number(p<0.01) compared to that achieved by DHEA alone (FIG. 7). Similarly,the addition of EM-800 to DHEA treatment, resulted in an additional 15%(p<0.05) decrease in trabecular bone separation, compared to thatachieved with DHEA alone, thus leading to values not different fromthose seen in intact controls.

As complement to the numerical data presented in FIGS. 6 and 7, FIG. 8illustrates the increase in trabecular bone volume in the proximal tibiametaphysis induced by DHEA in ovariectomized treated animals (C)compared to ovariectomized controls (B), as well as the partialinhibition of the stimulatory effect of DHEA after the addition ofFlutamide to DHEA treatment (D). On the other hand, administration ofDHEA in combination with EM-800 resulted in a complete prevention of theovariectomy-induced osteopenia (E), the trabecular bone volume beingcomparable to that seen in intact controls (A).

The bone loss observed at menopause in women is believed to be relatedto an increase in the rate of bone resorption which is not fullycompensated by the secondary increase in bone formation. In fact, theparameters of both bone formation and bone resorption are increased inosteoporosis and both bone resorption and formation are inhibited byestrogen replacement therapy. The inhibitory effect of estrogenreplacement on bone formation is thus believed to result from a coupledmechanism between bone resorption and bone formation, such that theprimary estrogen-induced reduction in bone resorption entrains areduction in bone formation (Parfitt, Calcified Tissue International 36Suppl. 1: S37-S45, 1984).

Cancellous bone strength and subsequent resistance to fracture do notonly depend upon the total amount of cancellous bone but also on thetrabecular microstructure, as determined by the number, size, anddistribution of the trabeculae. The loss of ovarian function inpostmenopausal women is accompanied by a significant decrease in totaltrabecular bone volume (Melsen et al., Acta Pathologica & MicrobiologicaScandinavia 86: 70-81, 1978; Vakamatsou et al., Calcified TissueInternational 37: 594-597, 1985), mainly related to a decrease in thenumber and, to a lesser degree, in the width of trabeculae (Weinsteinand Hutson, Bone 8: 137-142, 1987).

In the present study, the androgenic stimulatory effect of DHEA wasobserved on almost all the bone histomorphometric parameters studied.DHEA thus resulted in a significant increase in trabecular bone volumeas well as trabecular number, while it decreased the intertrabeculararea.

In order to facilitate the combination therapy aspect of the invention,for any indication discussed herein, the invention contemplatespharmaceutical compositions which include both the SERM or thebisphosphonate compound and the sex steroid precursor (DHEA, DHEAS,5-diol) in a single composition for simultaneous administration. Thecomposition may be suitable for administration in any traditional mannerincluding but not limited to oral administration, subcutaneousinjection, intramuscular injection or percutaneous administration. Inother embodiments, a kit is provided wherein the kit includes one ormore SERM or bisphosphonate and sex steroid precursors in separate or inone container. The kit may include appropriate materials for oraladministration, e.g. tablets, capsules, syrups and the like and fortransdermal administration, e.g., ointments, lotions, gels, creams,sustained release patches and the like.

Applicants believe that administration of SERMs and sex steroidprecursors has utility in the treatment and/or prevention of thedevelopment of osteoporosis, breast cancer, hypercholesterolemia,hyperlipidemia or atherosclerosis. The active ingredients of theinvention (whether SERM or precursor or bisphosphonate or otherwise) maybe formulated and administered in a variety of manner.

Active ingredient for transdermal or transmucosal is preferably presentat from 0.5% to 20% by weight relative to the total weight of thepharmaceutical composition more preferably between 2 and 10%. DHEA or5-diol should be at a concentration of at least 7% for percutaneousadministration. Alternatively, the active ingredient may be placed intoa transdermal patch having structures known in the art, for example,structures such as those set forth in E.P. Patent No. 0279982.

When formulated as an ointment, lotion, gel or cream or the like, theactive compound is admixed with a suitable carrier which is compatiblewith human skin or mucosa and which enhances transdermal penetration ofthe compound through the skin or mucosa. Suitable carriers are known inthe art and include but are not limited to Klucel HF and Glaxal base.Some are commercially available, e.g., Glaxal base available from GlaxoCanada Limited Company. Other suitable vehicles can be found in Kollerand Buri, S.T.P. Pharma 3(2), 115-124, 1987. The carrier is preferablyone in which the active ingredient(s) is (are) soluble at ambienttemperature at the concentration of active ingredient that is used. Thecarrier should have sufficient viscosity to maintain the inhibitor on alocalized area of skin or mucosa to which the composition has beenapplied, without running or evaporating for a time period sufficient topermit substantial penetration of the precursor through the localizedarea of skin or mucosa and into the bloodstream where it will cause adesirable clinical effect. The carrier is typically a mixture of severalcomponents, e.g. pharmaceutically acceptable solvents and a thickeningagent. A mixture of organic and inorganic solvents can aid hydrophylicand lipophylic solubility, e.g. water and an alcohol such as ethanol.

Preferred sex steroid precursors are dehydroepiandrosterone (DHEA)(available from Diosynth Inc., Chicago, Ill., USA), its prodrugs(available from Steraloids, Wilton, N.H., USA), 5-androsten-3β,17β-dioland its prodrugs EM-1304 and EM-01474-D (available from Steraloids,Wilton, N.H. USA).

It is preferred that the sex steroid precursor is formulated as analcoholic gel containing 2.0 to 10% of caprylic-capric triglyceride(Neobee M-5); 10 to 20% of hexylene glycol; 2.0 to 10% ofdiethyleneglycol monomethyl ether (Transutol); 2.0 to 10% ofCyclomethicone (Dow Corning 345); 1.0 to 2% of benzyl alcohol and 1.0 to5.0% of hydroxypropylcellulose (Klucel HF).

The carrier may also include various additives commonly used inointments and lotions and well known in the cosmetic and medical arts.For example, fragrances, antioxidants, perfumes, gelling agents,thickening agents such as carboxymethylcellulose, surfactants,stabilizers, emollients, coloring agents and other similar agents may bepresent. When used to treat systemic diseases, the site of applicationon the skin should be changed in order to avoid excess localconcentration of active ingredient and possible overstimulation of theskin and sebaceous glands by androgenic metabolites of sex steroidprecursor.

In a pharmaceutical composition for oral administration, DHEA or otherprecursor is preferably present in a concentration between 5 and 98% byweight relative to total weight of the composition more preferablybetween 50 and 98 percent, especially between 80 and 98 percent. Asingle precursor such as DHEA may be the only active ingredient, oralternatively, a plurality of precursors and/or their analogues may beused (e.g., a combination of DHEA, DHEA-S, 5-diol, or a combination oftwo or more compounds converted in vivo to DHEA, DHEA-S or 5-diol or acombination of DHEA or 5-diol and one or more analogues thereof whichare converted to DHEA or 5-diol in vivo, etc. The blood level of DHEA isthe final criteria of adequate dosage which takes into accountindividual variation in absorption and metabolism.

Preferably, the attending clinician will, especially at the beginning oftreatment, monitor an individual patient's overall response and serumlevels of DHEA (in comparison to the preferred serum concentrationsdiscussed above), and monitor the patient's overall response totreatment, adjusting dosages as necessary where a given patients'metabolism or reaction to treatment is atypical.

Treatment in accordance with the invention is suitable for indefinitecontinuation. It is expected that DHEA and/or 5-diol treatment willsimply maintain DHEA levels within a range similar to that which occursnaturally in women before menopause (serum concentration between 4 and10 micrograms per liter), or naturally in young adult men (serumconcentration between 4 and 10 micrograms per liter).

The SERM compound or bisphosphonate and/or the sex steroid precursor canalso be administered, by the oral route, and may be formulated withconventional pharmaceutical excipients, e.g. spray dried lactose,microcrystalline cellulose, and magnesium stearate into tablets orcapsules for oral administration.

The active substance can be worked into tablets or dragee cores by beingmixed with solid, pulverulent carrier substances, such as sodiumcitrate, calcium carbonate or dicalcium phosphate, and binders such aspolyvinyl pyrrolidone, gelatin or cellulose derivatives, possibly byadding also lubricants such as magnesium stearate, sodium laurylsulfate, “Carbowax” or polyethylene glycol. Of course, taste-improvingsubstances can be added in the case of oral administration forms.

As further forms, one can use plug capsules, e.g. of hard gelatin, aswell as closed solf-gelatin capsules comprising a softner orplasticizer, e.g. glycerine. The plug capsules contain the activesubstance preferably in the form of granulate, e.g. in mixture withfillers, such as lactose, saccharose, mannitol, starches, such as potatostarch or amylopectin, cellulose derivatives or highly dispersed silicicacids. In solf-gelatin capsules, the active substance is preferablydissolved or suspended in suitable liquids, such as vegetable oils orliquid polyethylene glycols.

The lotion, ointment, gel or cream should be thoroughly rubbed into theskin so that no excess is plainly visible, and the skin should not bewashed in that region until most of the transdermal penetration hasoccurred preferably at least 4 hours and, more preferably, at least 6hours.

A transdermal patch may be used to deliver precursor in accordance withknown techniques. It is typically applied for a much longer period,e.g., 1 to 4 days, but typically contacts active ingredient to a smallersurface area, allowing a slow and constant delivery of activeingredient.

A number of transdermal drug delivery systems that have been developed,and are in use, are suitable for delivering the active ingredient of thepresent invention. The rate of release is typically controlled by amatrix diffusion, or by passage of the active ingredient through acontrolling membrane.

Mechanical aspects of transdermal devices are well known in the rat, andare explained, for example, in U.S. Pat. Nos. 5,162,037, 5,154,922,5,135,480, 4,666,441, 4,624,665, 3,742,951, 3,797,444, 4,568,343,5,064,654, 5,071,644, 5,071,657, the disclosures of which areincorporated herein by reference. Additional background is provided byEuropean Patent 0279982 and British Patent Application 2185187.

The device may be any of the general types known in the art includingadhesive matrix and reservoir-type transdermal delivery devices. Thedevice may include drug-containing matrixes incorporating fibers whichabsorb the active ingredient and/or carrier. In a reservoir-type device,the reservoir may be defined by a polymer membrane impermeable to thecarrier and to the active ingredient.

In a transdermal device, the device itself maintains active ingredientin contact with the desired localized skin surface. In such a device,the viscosity of the carrier for active ingredient is of less concernthan with a cream or gel. A solvent system for a transdermal device mayinclude, for example, oleic acid, linear alcohol lactate and dipropyleneglycol, or other solvent systems known in the art. The active ingredientmay be dissolved or suspended in the carrier.

For attachment to the skin, a transdermal patch may be mounted on asurgical adhesive tape having a hole punched in the middle. The adhesiveis preferably covered by a release liner to protect it prior to use.Typical material suitable for release includes polyethylene andpolyethylene-coated paper, and preferably silicone-coated for ease ofremoval. For applying the device, the release liner is simply peeledaway and the adhesive attached to the patient's skin. In U.S. Pat. No.5,135,480, the disclosure of which is incorporated by reference, Bannonet al. describe an alternative device having a non-adhesive means forsecuring the device to the skin.

The percutaneous or transmucosal delivery system of the invention mayalso be used as a novel and improved delivery system for the preventionand/or treatment of osteoporosis or other diseases which respondfavorably to treatment with androgens and/or estrogens.

A selective estrogen receptor modulator of the invention has a molecularformula with the following features: a) two aromatic rings spaced by 1to 2 intervening carbon atoms, both aromatic rings being eitherunsubstituted or substituted by a hydroxyl group or a group converted invivo to hydroxyl; and b) a side chain possessing an aromatic ring and atertiary amine function or salt thereof.

One preferred SERM of the invention is EM-800 reported in PCT/CA96/00097(WO 96/26201) The molecular structure of EM-800 is:

Another preferred SERM of the invention is EM-01538:

EM-1538, (also called EM-652.HCL) is the hydrochloride salt of thepotent antiestrogen EM-652 compared to EM-800, EM-1538 is a simpler andeasier salt to synthesize. It was also easy to isolate, purify,crystallizable, and displayed good solid state stability. Inadministering either EM-800 or EM-1538, it is believed to result in thesame active compound in vivo.

Other preferred SERMs of the invention include Tamoxifen((Z)-2-[4-(1,2-diphenyl-1-butenylphenoxy)]-N,N-dimethylethanamine)(available from Zeneca, UK), Toremifene (available from Orion-FarmosPharmaceuticla, Finland, or Schering-Plough), Droloxifene and CP-336,156(cis-1R-[4′-pyrrolidino-ethoxyphenyl]-2S-phenyl-6-hydroxy-1,2,3,4,-tetrahydronapthaleneD-(−)-tartrate salt) (Pfizer Inc., USA), Raloxifene (Eli Lilly and Co.,USA), LY 335563 and LY 353381 (Eli Lilly and Co., USA), Iodoxifene(SmithKline Beecham, USA), Levormeloxifene(3,4-trans-2,2-dimethyl-3-phenyl-4-[4-(2-(2-(pyrrolidin-1-yl)ethoxy)phenyl]-7-methoxychroman)(Novo Nordisk, A/S, Denmark) which is disclosed in Shalmi et al. WO97/25034, WO 97/25035, WO 97/25037, WO 97/25038; and Korsgaard et al. WO97/25036), GW5638 (described by Willson at al., Endocrinology, 138(9),3901-3911, 1997) and indole derivatives (disclosed by Miller et al. EP0802183A1) and TSE 424 developed by Wyeth Ayers (USA) and disclosed inJP10036347 (American home products corporation) and nonsteroidalestrogen derivatives described in WO 97/32837.

Any SERM used as required for efficacy, as recommended by themanufacturer, can be used. Appropriate dosages are known in the art. Anyother non steroidal antiestrogen commercially available can be usedaccording to the invention. Any compound having activity similar toSERMs (example: Raloxifene can be used).

SERMs administered in accordance with the invention are preferablyadministered in a dosage range between 0.01 to 10 mg/kg of body weightper day (preferably 0.05 to 1.0 mg/kg), with 5 mg per day, especially 10mg per day, in two equally divided doses being preferred for a person ofaverage body weight when orally administered, or in a dosage rangebetween 0.003 to 3.0 mg/kg of body weight per day (preferably 0.015 to0.3 mg/ml), with 1.5 mg per day, especially 3.0 mg per day, in twoequally divided doses being preferred for a person of average bodyweight when parentally administered (i.e. intramuscular, subcutaneous orpercutaneous administration). Preferably the SERMs are administeredtogether with a pharmaceutically acceptable diluent or carrier asdescribed below.

Preferred bisphosphonates of the invention include Alendronate[(4-amino-1-hydroxybutylidene)bis phosphonic acid, disodium salt,hydrate] available from Merck Shape and Dohme under the Tradename ofFosamax, Etidronate [(1-hydroxyethylidene)bis phosphonic acid,2,2′-iminobis ethanol] available from Procter and Gamble under the Tradenames of Didrocal and Didronel, Clodronate [(dichloromethylene)bisphosphonic acid, disodium salt] available from Rhône-Poulenc Rorer underthe Trade name of Bonefos or available from Boehringer Mannheim underthe Trade name of Ostac and, Pamidronate(3-amino-1-hydroxypropylidene)bis phosphonic acid, disodium salt)available from Geigy under the Tradename of Aredia. Risedronate(1-hydroxy-2-(3-pyridinyl)ethylidene bisphosphonic acid monosodium salt)is under clinical development. Any other bisphosphonates commerciallyavailable can be used according to the invention, all at themanufacturers' recommended dosage. Likewise sex steroid precursors maybe utilized at dosages recommended in the prior art, preferably atdosages that restore circulating levels to those of healthy males 20-30years of age or those of premenopausal adult females.

With respect to all of the dosages recommended herein, the attendingclinician should monitor individual patient response and adjust dosageaccordingly.

EXAMPLES Example 1

Materials ad Methods

Animals

Female Sprague-Dawley rats [Crl:CD(SD)Br] were obtained at 44-46 days ofage from Charles River Canada Inc. (St. Constant, Quebec) and housed 2per cage in a light (12 h light/day; lights on at 07:15 h)—andtemperature (22±2° C.)—controlled environment. Animals received Purinarodent chow and tap water ad libitum. The animal studies were conductedin a Canadian Council on Animal Care (CCAC)-approved facility inagreement with the CCAC Guide for Care and Use of Experimental Animals.

Induction of Mammary Tumors by DMBA

Mammary carcinomas were induced by a single intragastric administrationof 20 mg of DMBA (Sigma Chemical Co., St. Louis, Mo.) in 1 ml of cornoil at 50-52 days of age. Two months later, tumor measurement wasperformed biweekly. The two largest perpendicular diameters of eachtumor were recorded with calipers to estimate tumor size as described(Asselin et al., Endocrinology 101: 666-671, 1977). Tumor site, size andnumber were recorded.

Treatment

The animals were randomly divided into groups each containing 20 ratswith the exception of 40 animals in the control group. The animals weretreated for 282 days with the following: (1) control vehicles, for bothDHEA and EM-800; (2) EM-800((+)-7-pivaloyloxy-3-(4′-pivaloyloxyphenyl)-4-methyl-2-(4″-(2′″-piperidinoethoxy)phenyl)-2H-benzopyran)(75 μg, orally, once daily) in 0.5 ml of a 4% ethanol, 4% polyethyleneglycol-600, 1% gelatin, 0.9% NaCl suspension; (3) DHEA (10 mg,percutaneously, once daily) in 0.5 ml of 50% ethanol, 50% propyleneglycol; and (4) both EM-800 and DHEA. Treatment was initiated 3 daysbefore the oral administration of DMBA. EM-800 was synthesized in theMedicinal Chemistry Division of our laboratory while DHEA was purchasedfrom Steraloids Inc., Wilton, N.H.

Many of the control animals and some of EM-800- or DHEA-treated animalswere sacrificed by cervical dislocation under isoflurane-inducedanesthesia 6 months after DMBA administration because of the too largesize of tumors. The values of tumor size and number of these rats atsacrifice, together with those measured at later time intervals from thesurviving animals, were used for the later analysis of the incidence oftumors, average tumor number per tumor-bearing rat and average tumorsize per tumor-bearing animal. The remaining animals (9 rats fromcontrol and 13-19 rats from each other group) continued to receivetreatment for another 3-month period in order to observe long-termpreventive potency of DHEA and EM-800 alone or in combination. Rats weresacrificed 279 days after DMBA administration. The uteri, vaginas, andovaries were immediately removed, freed from connective and adiposetissue, and weighed.

Sample Collection and Processing

Twenty-four-hour urinary samples were collected at the end of theexperiment from the first 9 rats of each group following transfer inmetabolic cages (Allentown Caging Equipment Co., Allentown, N.J.). Twourinary samples were collected and analyzed on different days for eachanimal in order to minimize the influence of daily variation. Therefore,each value shown represents the mean of the two measurements performedon two different days. 0.5 ml of toluene was added into the urinecollecting tubes to prevent urine evaporation and bacterial growth andthe urinary volume was recorded. Trunk blood was collected at sacrificeand was allowed to clot at 4° C. overnight before centrifugation at 3000rpm for 30 min.

Analysis of Urine and Serum Biochemical Parameters

Fresh samples were used for the assay of urinary creatinine, calcium,and phosphorus as well as serum total alkaline phosphatase (tALP)activity, cholesterol and triglycerides. These biochemical parameterswere measured automatically with a Monarch 2000 Chemistry System(Instrumentation Laboratory Co. Lexington, Mass.) under Good LaboratoryPractice conditions. Urinary hydroxyproline was measured as described(Podenphant et al., Clinica Chimica Acta 142: 145-148, 1984).

Bone Mass Measurements

Rats were anesthetized with an i.p. injection of ketamine hydrochlorideand diazepam at doses of 50 and 4 mg/kg B.W., respectively. The wholeskeleton and the right femur were scanned using dual energy X-rayabsorptiometry (DEXA; QDR 2000-7.10 C, Hologic, Waltham, Mass.) equippedwith a Regional High Resolution software. The scan field sizes were28.110×17.805 cm and 5.0×1.902 cm, the resolutions were 0.1511×0.0761 cmand 0.0254×0.0127 cm, while the scan speeds were 0.3608 and 0.0956mm/sec for total skeleton and femur, respectively. Both bone mineralcontent (BMC) and bone mineral density (BMD) of total skeleton, lumbarspine, and femur were measured on the scan images of total skeleton andfemur.

Statistical Analyses

Statistical significance was measured according to the multiple rangetest of Duncan-Kramer (Biometrics 12: 307-310, 1956). Analysis of theincidence of development of mammary tumors was performed using theFisher's exact test (Conover, Practical nonparametric statistics, 2ndEdition 153-170, 1980). The data are presented as means±S.E.M.

Results

Effect on the Development of DMBA-Induced Mammary Carcinoma

As illustrated in FIG. 1, 95% of control animals developed palpablemammary tumors by 279 days after DMBA administration. Treatment withDHEA or EM-800 partially prevented the development of DMBA-inducedmammary carcinoma and the incidence was thus reduced to 57% (p<0.01) and38% (p<0.01), respectively. Interestingly, combination of the twocompounds led to a significantly higher inhibitory effect than thoseachieved by each compound alone (p<0.01 versus DHEA or EM-800 alone). Infact, the only two tumors which developed in the group of animalstreated with both compounds disappeared before the end of experiment.

Treatment with DHEA or EM-800 decreased average tumor number pertumor-bearing animal from 4.7±0.5 tumors in control animals to 3.4±0.7(N.S.) and 1.4±0.3 (p<0.01) tumors/animal, respectively, while no tumorwas found at the end of the experiment in the animals who received bothdrugs (p<0.01 versus the three other groups) (FIG. 2A). One of the twotumors which later disappeared was present from day 79 to day 201following DMBA administration while the other tumor was palpable fromday 176 to day 257. It can be seen in FIG. 2B that DHEA or EM-800 alonedecreased average tumor area per tumor-bearing animal from 12.8±1.3 cm²at the end of the experiment to 10.2±2.1 cm² (N.S.) and 7.7±1.8 cm²(N.S.), respectively, while the combination treatment resulted in a zerovalue (p<0.01 versus the three other groups). The two tumors whichdeveloped in the group of animals treated with both DHEA and EM-800 didnot grow larger than 1 cm². It should be mentioned that the real valuesof average tumor area as well as the average tumor number pertumor-bearing animal in the control group should be higher than thevalues presented in FIG. 2, since many rats had to be sacrificed beforethe end of the experiment because of the excessive size of tumors. Thevalues measured at time of sacrifice were thus included as such in thecalculations made at later time intervals in order to minimize a bias inthe control group which, in any case, remained significantly above theother groups.

Effect on Bone

Long-term percutaneous administration of DHEA to female rats induced6.9% (p<0.01), 10.6% (p<0.05), and 8.2% (p<0.01) increases in bonemineral density (BMD) of total skeleton, lumbar spine, and femur,respectively (Table 2). On the other hand, no significant change wasfound in the animals treated with EM-800. Furthermore, when bothcompounds were administered simultaneously, the values obtained werecomparable to those achieved with DHEA alone.

Treatment with DHEA increased serum total alkaline phosphatase (tALP)activity by 74% (p<0.05), but had no effect on daily urinary calcium andphosphorus excretion and on the urinary ratio of hydroxyproline tocreatinine (Table 3). On the other hand, treatment with EM-800 decreasedthe urinary hydroxyproline to creatinine ratio by 48% (p<0.01), but hadno statistically significant influence on daily urinary calcium orphosphorus excretion and serum tALP activity. The combination of DHEAand EM-800 led to an increase in serum tALP activity (p<0.01) similar tothat achieved with DHEA alone and reduced the urinary hydroxyproline tocreatinine ratio by 69%, a value significantly (p<0.01) lower than thatachieved with EM-800 alone. In addition, the combination of the twodrugs significantly reduced daily urinary calcium and phosphorusexcretion by 84% (p<0.01) and 56% (p<0.01), respectively, while nosignificant change was observed with each drug alone (Table 3).

Effect on Serum Lipid Levels

Long-term treatment with EM-800 lowered serum triglyceride andcholesterol levels by 72% (p<0.01) and by 45% (p<0.01), respectively,whereas long-term administration of DHEA decreased serum triglycerideslevels by 60% (p<0.01), serum cholesterol levels being unaffected.Moreover, 42% (p<0.01) and 52% (p<0.01) decreases in serum triglycerideand cholesterol concentrations were measured in the animals treated withboth EM-800 and DHEA (FIG. 3).

TABLE 2 Effect of treatment with DHEA (10 mg, percutaneously, oncedaily) or EM-800 (75 μg, orally, once daily) alone or in combination for9 months on bone mineral density (BMD) of femur, lumbar spine, and totalskeleton in the female rat. BMD (g/cm2) GROUP TOTAL SKELETON LUMBARSPINE FEMUR CONTROL 0.1371 ± 0.0025 0.1956 ± 0.0067 0.3151 ± 0.0063 DHEA(10 mg) 0.1465 ± 0.0010** 0.2163 ± 0.0049* 0.3408 ± 0.0038** EM-800 (75μg) 0.1356 ± 0.0017 0.1888 ± 0.0045 0.3097 ± 0.0047 DHEA + EM-800 0.1498± 0.0019** 0.2108 ± 0.0061 0.3412 ± 0.0056** Measurements were performedin 9 rats per group. *p < 0.05; **p < 0.01, experimental versus control.

TABLE 3 Effect of treatment with DHEA (10 mg, percutaneously, oncedaily) or EM-800 (75 μg, orally, once daily) alone or in combination for9 months on parameters of bone metabolism in the rat: daily urinarycalcium and phosphorus excretion, urinary hydroxyproline to creatinineratio (HP/Cr), and serum total alkaline phosphotase activity (tALP).URINE CALCIUM PHOSPHORUS HP/Cr SERUM tALP GROUP (μmol/24 h/100 g)(μmol/24 h/100 g) (μmol/mmol) (IU/L) CONTROL 23.17 ± 1.55 132.72 ± 6.0813.04 ± 2.19 114.25 ± 14.04 DHEA (10 mg) 25.87 ± 3.54 151.41 ± 14.5714.02 ± 1.59 198.38 ± 30.76* EM-800 (75 μg) 17.44 ± 4.5 102.03 ± 25.13 6.81 ± 0.84** 114.11 ± 11.26 DHEA + EM-800  3.71 ± 0.75**  59.06 ±4.76**  4.06 ± 0.28** 204.38 ± 14.20** Samples were obtained from 9animals per group. *p < 0.05; **p < 0.01 experimental versus control.

Example 2

Abstract

In the mammary gland, androgens are formed from the precursor steroiddehydroepiandrosterone (DHEA). Clinical evidence indicates thatandrogens have inhibitory effects on breast cancer. Estrogens, on theother hand, stimulate the development and growth of breast cancer. Westudied the effect of DHEA alone or in combination with the newlydescribed pure antiestrogen, EM-800, on the growth of tumor xenograftsformed by the human breast cancer cell line ZR-75-1 in ovariectomizednude mice.

Mice received daily subcutaneous injections of 0.5 μg estrone (anestrogenic hormone) immediately after ovariectomy. EM-800 (15, 50 or 100μg) was given orally once daily. DHEA was applied twice daily (totaldose 0.3, 1.0 or 3.0 mg) to the dorsal skin either alone or incombination with a 15 μg daily oral dose of EM-800. Changes in tumorsize in response to the treatments were assessed periodically inrelation to the measurements made on the first day. At the end of theexperiments, tumors were dissected and weighed.

A 9.4-fold increase in tumor size in 9.5 months was observed inovariectomized mice receiving estrone alone in comparison with mice notreceiving estrone. Administration of 15, 50 or 100 μg EM-800 inestrone-supplemented ovariectomized led to inhibitions of 88%, 93%, and94% in tumor size, respectively. DHEA, on the other hand, at doses of0.3, 1.0 or 3.0 mg inhibited terminal tumor weight by 67%, 82%, and 85%,respectively. Comparable inhibitions in tumor size were obtained with adaily 15 μg oral dose of EM-800 with or without different doses ofpercutaneous DHEA.

DHEA and EM-800 independently suppressed the growth ofestrone-stimulated ZR-75-1 mouse xenograft tumors in nude mice.Administration of DHEA at the defined doses does not alter theinhibitory effect of EM-800.

Materials and Methods

ZR-75-1 Cells

ZR-75-1 human breast cancer cells were obtained from the American TypeCulture Collection (Rockville, Md.) and routinely cultured as monolayersin RPMI 1640 medium supplemented with 2 mM L-glutamine, 1 mM sodiumpyruvate, 100 IU penicillin/ml, 100 μg streptomycin/ml, and 10% fetalbovine serum, under a humidified atmosphere of 95% air/5% CO₂ at 37° C.as described (Poulin and Labrie, Cancer Res. 46: 4933-4937, 1986; Poulinet al., Breast Cancer Res. Treat. 12: 213-225, 1988). Cells werepassaged weekly after treatment with 0.05% trypsin:0.02% EDTA (w/v). Thecell cultures used for the experiments described in this report werederived from passage 93 of the cell line ZR-75-1.

Animals

Female homozygous Harlan Sprague-Dawley (nu/nu) athymic mice (28- to42-day-old) were obtained from HSD (Indianapolis, Ind., USA). Mice werehoused in vinyl cages with air filter tops in laminar air flow hoods andmaintained under pathogen-limited conditions. Cages, bedding, and foodwere autoclaved before use. Water was autoclaved, acidified to pH 2.8,and provided ad libitum.

Cell Inoculation

Mice were bilaterally ovariectomized (OVX) one week before tumor cellinoculation under anesthesia achieved by intraperitoneal injection of0.25 ml/animal of Avertin (amylic alcohol: 0.8 g/100 ml 0.9% NaCl; andtribromo ethanol: 2 g/100 ml 0.9% NaCl). 1.5×10⁶ ZR-75-1 cells inlogarithmic growth phase were harvested after the treatment of monolayerwith 0.05% trypsin/0.02% EDTA (w/v), were suspended in 0.1 ml of culturemedium containing 25% Matrigel and were inoculated subcutaneously onboth flanks of the animals using a 1 inch-long 20-gauge needle asdescribed previously (Dauvois et al., Cancer Res. 51: 3131-3135, 1991).In order to facilitate growth of the tumors, each animal received dailysubcutaneous injection of 10 μg of estradiol (E₂) in vehicle composed of0.9% NaCl 5% ethanol 1% gelatin for 5 weeks. After appearance ofpalpable ZR-75-1 tumors, tumor diameter was measured with calipers andmice having tumor diameter between 0.2 and 0.7 cm were selected for thisstudy.

Hormonal Treatment

All animals, except those in the control OVX group, received dailysubcutaneous injections of 0.5 μg estrone (E₁) in 0.2 ml of 0.9% NaCl 5%ethanol 1% gelatin. In the indicated groups, DHEA was administeredpercutaneously twice daily at the doses of 0.3, 1.0 or 3.0 mg/animalapplied in a volume of 0.02 ml on the dorsal skin area outside the areaof tumor growth. DHEA was dissolved in 50% ethanol 50% propylene glycol.EM-800,((+)-7-pivaloyloxy-3-(4′-pivaloyloxyphenyl)-4-methyl-2-(4″-(2′″-piperidinoethoxy)phenyl)-2H-benzopyran),was synthesized as described earlier (Gauthier et al., J. Med. Chem. 40:2117-2122, 1997) in the medicinal chemistry division of the Laboratoryof Molecular Endocrinology of the CHUL Research Center. EM-800 wasdissolved in 4% (v/v) ethanol 4% (v/v) polyethylene glycol (PEG) 600 1%(w/v) gelatin 0.9% (w/v) NaCl. Animals of the indicated groups receiveddaily oral doses of 15 μg, 50 μg, or 100 μg of EM-800 alone or incombination with DHEA while animals of the OVX group received thevehicle (0.2 ml 4% ethanol 4% PEG 600 1% gelatin 0.9% NaCl) alone.Tumors were measured once a week with Vernier calipers. Twoperpendicular diameters in cms (L and W) were recorded and tumor area(cm²) was calculated using the formula: L/2×W/2×π (Dauvois et al.,Cancer Res. 51: 3131-3135, 1991). The area measured on the first day oftreatment was taken as 100% and changes in tumor size were expressed aspercentage of initial tumor area. In case of subcutaneous tumors ingeneral, it is not possible to accurately access three dimensionalvolume of tumor, therefore, only tumors areas were measured. After 291days (or 9.5 months) of treatment, the animals were sacrificed.

The categories of responses were evaluated as described (Dauvois et al.,Breast Cancer Res. Treat. 14: 299-306, 1989; Dauvois et al., Eur. J.Cancer Clin. Oncol. 25: 891-897, 1989; Labrie et al., Breast Cancer Res.Treat. 33: 237-244, 1995). In short, partial regression corresponds tothe tumors that regressed equal to or more than 50% of their originalsize; stable response refers to tumors that regressed less than 50% ofthe original size or progressed less than 50% of their original size,while complete regression refers to those tumors that were undetectableat the end of treatment. Progression refers to tumors that progressedmore than 50% compared with their original size. At the end of theexperiment, all animals were killed by decapitation. Tumors, uterus, andvagina were immediately removed, freed from connective and adiposetissues, and weighed.

Statistical Analysis

Statistical significance of the effects of treatments on tumor size wasassessed using an analysis of variance (ANOVA) evaluating the effectsdue to DHEA, EM-800, and time, and repeated measures in the same animalsperformed at the initiation and at the end of the treatment (subjectswithin group factor). The repeated measures at time 0 and after 9.5months of treatment constitute randomized blocks of animals. The time isthus analyzed as a within-block effect while both treatments areassessed as between-block effects. All interactions between main effectswere included in the model. The significance of the treatment factorsand of their interactions was analyzed using the subjects within groupas the error term. Data were log-transformed. The hypotheses underlyingthe ANOVA assumed the normality of the residuals and the homogeneity ofvariance.

A posteriori pairwise comparisons were performed using Fisher's test forleast significant difference. Main effects and the interaction oftreatments on body weight and organ weight were analyzed using astandard two-way ANOVA with interactions. All ANOVAs were performedusing SAS program (SAS Institute, Cary, N.C., USA). Significance ofdifferences were declared using a 2-tailed test with an overall level of5%.

Categorical data were analyzed with a Kruskall-Wallis test for orderedcategorical response variables (complete response, partial response,stable response, and progression of tumor). After overall assessment ofa treatment effects, subsets of the results presented in Table 4 wereanalyzed adjusting the critical p-value for multiple comparisons. Theexact p-values were calculated using StatXact program (Cytel, Cambridge,Mass., USA).

Data are expressed as means± standard error of the mean (SEM) of 12 to15 mice in each group.

Results

As illustrated in FIG. 4A, human ZR-75-1 tumors increased by 9.4-foldover 291 days (9.5 months) in ovariectomized nude mice treated with adaily 0.5 μg subcutaneously administered dose of estrone while incontrol OVX mice who received the vehicle alone, tumor size wasdecreased to 36.9% of the initial value during the course of the study.

Treatment with increasing doses of percutaneous DHEA caused aprogressive inhibition of E₁-stimulated ZR-75-1 tumor growth.Inhibitions of 50.4%, 76.8%, and 80.0% were achieved at 9.5 months oftreatment with the 0.3 mg, 1.0 mg, and 3.0 mg daily doses per animal ofDHEA, respectively (FIG. 4A). In agreement with the decrease in totaltumor load, treatment with DHEA led to a marked decrease of the averageweight of the tumors remaining at the end of the experiment. In fact,average tumor weight decreased from 1.12±0.26 g in controlE₁-supplemented ovariectomized nude mice to 0.37±0.12 g (P=0.005),0.20±0.06 g (P=0.001), and 0.17±0.06 g (P=0.0009) in the groups ofanimals receiving the daily 0.3, 1.0 and 3.0 mg doses of DHEA,respectively (FIG. 4B).

At the daily doses of 15 μg, 50 μg, and 100 μg, the antiestrogen EM-800inhibited estrogen-stimulated tumor size by 87.5% (P<0.0001), 93.5%(P<0.0001), and 94.0% (P=0.0003), respectively (FIG. 5A) when comparedto the tumor size in control animals at 9.5 months. The tumor sizereductions achieved with the three EM-800 doses are not significantlydifferent between each other. As illustrated in FIG. 4B, tumor weight atthe end of the 9.5-month study was decreased from 1.12±0.26 g in controlE₁-supplemented OVX mice to 0.08±0.03 g, 0.03±0.01 g and 0.04±0.03 g inanimals treated with the daily 15 μg, 50 μg, and 100 μg doses of EM-800,respectively (P<0.0001 at all doses of EM-800 vs E₁ supplemented OVX).

As mentioned above, the antiestrogen EM-800, at the daily oral dose of15 μg, caused a 87.5% inhibition of estrone-stimulated tumor growthmeasured at 9.5 months. The addition of DHEA at the three doses used hadno significant effect on the already marked inhibition of tumor sizeachieved with the 15 μg daily dose of the antiestrogen EM-800 (FIG. 5B).Thus, average tumor weight was dramatically reduced from 1.12±0.26 g incontrol estrone-supplemented mice to 0.08±0.03 g (P<0.0001), 0.11±0.04 g(P=0.0002), 0.13±0.07 g (P=0.0004) and 0.08±0.05 g (P<0.0001) in theanimals who received the daily dose of 15 μg of the antiestrogen aloneor in combination with the 0.3, 1.0, and 3.0 mg doses of DHEA,respectively (no significant difference was noted between the 4 groups)(FIG. 4B).

It was also of interest to examine the categories of responses achievedwith the above-indicated treatments. Thus, treatment with the increasingdoses of DHEA decreased, although not to a level of statisticalsignificance (P=0.088), the number of progressing tumors from 87.5% inthe control OVX animals supplemented with estrone to values of 50.0%,53.3%, and 66.7% in the animals treated with the daily doses of 0.3, 1.0or 3.0 mg of DHEA (Table 4). Complete responses, on the other hand,increased from 0% in the estrone-supplemented mice to 28.6%, 26.7%, and20.0% in the animals receiving the 0.3, 1.0, and 3.0 mg daily doses ofpercutaneous DHEA. Stable responses, on the other hand, were measured at12.5%, 21.4%, 20.0%, and 13.3% in the control E₁-supplemented mice andin the three groups of animals who received the above-indicated doses ofDHEA, respectively. In control ovariectomized mice, the rates ofcomplete, partial and stable responses were measured at 68.8%, 6.2%, and18.8%, respectively, while progression was seen in only 6.2% of tumors(Table 4).

Complete responses or disappearance of the tumors were achieved in29.4%, 33.3%, 26.7%, and 35.3% of tumors in the animals who received theantiestrogen EM-800 (P=0.0006) alone (15 μg) or in combination with the0.3 mg, 1.0 mg, or 3.0 mg of DHEA, respectively (Table 4). Progression,on the other hand, was seen in 35.3%, 44.4%, 53.3%, and 17.6% of thetumors, in the same groups of animals, respectively. There is nosignificant difference between the groups treated with EM-800, eitheralone or in combination with DHEA.

No significant effect of DHEA or EM-800 treatment was observed on bodyweight adjusted for tumor weight. Treatment of OVX mice with estrone,increased uterine weight from 28±5 mg in OVX control mice to 132±8 mg(P<0.01) while increasing doses of DHEA caused a progressive butrelatively small inhibition of the stimulatory effect of estrone whichreached 26% (P=0.0008) at the highest dose of DHEA used. It can be seenin the same figure that estrone-stimulated uterine weight was decreasedfrom 132±8 mg in control estrone-supplemented mice to 49±3 mg, 36±2 mg,and 32±1 mg (P<0.0001 at all doses vs control) with the daily oral dosesof 15 μg, 50 μg, or 100 μg of EM-800 (overall P<0.0001), respectively.Fifteen micrograms (15 μg) EM-800 in combination with the 0.3 mg, 1.0 mgor 3.0 mg daily doses of DHEA, uterine weight was measured at 46±3 mg,59±5 mg and 69±3 mg, respectively.

On the other hand, treatment with estrone increased vaginal weight from14±2 mg in OVX animals to 31±2 mg (P<0.01) while the addition of DHEAhad no significant effect. Vaginal weight was then reduced to 23±1 mg,15±1 mg, and 11±1 mg following treatment with the daily 15 μg, 50 μg or100 μg doses of EM-800, respectively (overall p and pairwise P<0.0001 atall doses vs control). In combination with the 0.3 mg, 1.0 mg or 3.0 mgdoses of DHEA and of EM-800, vaginal weight was measured at 22±1 mg,25±2 mg and 23±1 mg, respectively (N.S. for all groups versus 15 μgEM-800). It should be mentioned that at the highest dose used, namely100 μg daily, EM-800 decreased uterine weight in estrone-supplementedOVX animals to a value not different from that of OVX controls whilevaginal weight was reduced to a value below that measured in OVXcontrols (P<0.05). DHEA, probably due to its androgenic effects,partially counteracted the effect of EM-800 on uterine and vaginalweight.

TABLE 4 Effect of percutaneous administration of DHEA or oraladministration of EM-800 alone or in combination for 9.5 months on theresponses (complete, partial, stable, and progression) of human ZR-75-1breast tumor xenografts in nude mice. TOTAL CATEGORY OF RESPONSE NUMBEROF Complete Partial Stable Progression GROUP ANIMALS Number and (%) OVX16 11 (68.8)    1 (6.2) 3 (18.8) 1 (6.2)  OVX + E1 (0.5 μg) 16 0 (0)   0(0) 2 (12.5) 14 (87.5)  OVX + E1 (0.5 μg) + DHEA 0.3 mg 14 4 (28.6) 0(0) 3 (21.4) 7 (50.0) 1.0 mg 15 4 (26.7) 0 (0) 3 (20.0) 8 (53.3) 3.0 mg15 3 (20.0) 0 (0) 2 (13.3) 10 (66.7)  OVX + E1 (0.5 μg) + 15 μg 17 5(29.4)   1 (5.9) 5 (29.4) 6 (35.3) EM-800 50 μg 16 4 (25.0)   3 (18.8) 5(31.2) 4 (25.0) 100 μg 16 8 (50.0) 0 (0) 3 (18.8) 5 (31.2) OVX + E1 (0.5μg) + EM- 0.3 mg 18 6 (33.3) 0 (0) 4 (22.2) 8 (44.4) 800 + DHEA 1.0 mg15 4 (26.7) 0 (0) 3 (20.0) 8 (53.3) 3.0 mg 17 6 (35.3) 0 (0) 8 (47.1) 3(17.6) E₁ = Estrone; DHEA = dehydroepiandrosterone; OVX = ovariectomized

Example 3 Effect of the Preferred Compound of the Invention onCholesterol Levels of Female Ovariectomized Rats

Animals and Treatment

Fifty to 60 day-old female Sprague-Dawley rats (Crl:CD(SD)Br) (CharlesRiver Laboratory, St-Constant, Canada) weighing approximately 190 g atthe time of ovariectomy were used. The animals were acclimated to theenvironmental conditions (temperature: 22±3° C.; humidity: 50±20%; 12-hlight-512-h dark cycles, lights on at 07:15 h) for 1 week before thesurgery. The animals were housed three per cage and were allowed freeaccess to tap water and a pelleted certified rodent feed (Lab Diet 5002,Ralston Purina, St-Louis, Mo.). The experiment was conducted in aCanadian Council on Animal Care approved facility in accordance with theCCAC Guide for Care and Use of Experimental Animals.

One hundred thirty-six female rats were ovariectomized under Isofluraneanesthesia on day 0 of the study and were randomly distributed into 17groups of animals to conduct the study outlined below:

Group 1: OVX CONT

Group 2: OVX+EM-800 (0.01 mg/kg, po, ID)

Group 3: OVX+EM-800 (0.03 mg/kg, po, ID)

Group 4: OVX+EM-800 (0.1 mg/kg, po, ID)

Group 5: OVX+EM-800 (0.3 mg/kg, po, ID)

Group 6: OVX+EM-800 (1 mg/kg, po, ID)

Group 7: OVX+EM-01538 (0.01 mg/kg, po, ID)

Group 8: OVX+EM-01538 (0.03 mg/kg, po, ID)

Group 9: OVX+EM-01538 (0.1 mg/kg, po, ID)

Group 10: OVX+EM-01538 (0.3 mg/kg, po, ID)

Group 11: OVX+EM-01538 (1 mg/kg, po, ID)

Group 12: OVX+Raloxifene (EM-1105) (0.01 mg/kg, po, ID)

Group 13: OVX+Raloxifene (EM-1105) (0.03 mg/kg, po, ID)

Group 14: OVX+Raloxifene (EM-1105) (0.1 mg/kg, po, ID)

Group 15: OVX+Raloxifene (EM-1105) (0.3 mg/kg, po, ID)

Group 16: OVX+Raloxifene (EM-1105) (1 mg/kg, po, ID)

Group 17: INT CONT

The administration of treatments were started on day 10 of the study andwere given by oral gavage once daily until day 13 of the study. Dosingsuspensions were prepared in 0.4% methylcellulose and the concentrationwas adjusted according to the mean body weight of the group recorded onday 10 of the study in order to give 0.5 ml of dosing suspension perrat. Approximately 24 hours after the last dosing, overnight fasteranimals were killed by exsanguination at the abdominal aorta underisoflurane anesthesia and blood samples were processed for serumpreparation. The uteri were removed, stripped of remaining fat andweighed.

Serum Cholesterol and Triglyceride Assays

Total serum cholesterol and triglyceride levels were determined usingthe Boehringer Mannheim Diagnostic Laboratory Systems).

Example 4

Androstene-3β,17β-diol (5-diol) possesses intrinsic estrogenic activity.In addition, as a precursor sex steroid, it can be transformed intoactive androgens and/or other estrogens in peripheral intracrinetissues. In order to assess the relative importance of the androgenicand estrogenic components of 5-diol action on bone mass, twenty-one weekold rats were ovariectomized and treated percutaneously once daily with2, 5, or 12.5 mg of 5-diol alone or in combination with the antiandrogenFlutamide (FLU, 10 mg, s.c., once daily), and/or the antiestrogen EM-800(100 μg, s.c., once daily) for 12 months. Bone mineral density (BMD) wasmeasured after 11 months of treatment. Ovariectomy (OVX) led to a 12.8%decrease in femoral BMD (p<0.01) while treatment with the highest doseof 5-diol restored 34.3% of femoral BMD lost during the 11 monthsfollowing OVX (p<0.01). Simultaneous administration of FLU completelyprevented the stimulatory effect of 5-diol on femoral BMD while theaddition of EM-800 resulted in an additional 28.4% stimulation comparedto the effect of 5-diol alone. The simultaneous administration of5-diol, FLU, and EM-800 only displayed the effect of EM-800 (27%) sincethe effect of 5-diol was completely blocked by FLU. Comparable resultswere obtained on BMD of lumbar spine although lumbar spine BMD in OVXrats receiving 12.5 mg 5-diol alone, 12.5 mg 5-diol+EM-800 or5-diol+FLU+EM-800 was restored to values not significantly differentfrom those of intact animals. The histomorphometric analysis shows thatthe stimulatory effects of 5-diol on bone volume, trabecular number andthe inhibitory effect on trabecular separation of secondary spongiosa ofthe proximal tibia metaphyseal area are abolished by FLU, but furtherenhanced by EM-800. The marked stimulation of serum alkaline phosphataseactivity obtained following the treatment with 5-diol is 57% (p<0.01 vs12.5 mg 5-diol alone) reversed by the simultaneous administration ofFLU. Treatment with 5-diol had no statistically significant inhibitoryeffect on the urinary ratio of calcium to creatinine. The highest doseof 5-diol caused a significant 23% (p<0.01) reduction of serumcholesterol while the addition of EM-800 decreased serum cholesterol by62% (p<0.01).

The present data clearly show the stimulatory effect of 5-diol on boneformation and suggest that although 5-diol is a weak estrogen, itsstimulatory effect on bone formation is predominantly mediated by anandrogenic effect. Moreover, the additive stimulatory effects of EM-800and 5-diol on bone mass demonstrate the bone-sparing effect of theantiestrogen EM-800 in the rat. The cholesterol-lowering activity ofboth 5-diol and EM-800 could have interesting utility for the preventionof cardiovascular diseases.

Example 5

Serum Urinary OH- GnRH Alkaline proline/ mRNA levels phosphatasecreatinin LH silver grains Cholesterol Triglycerides Group IU/Lμmol/mmol ng/ml per cell mmol/L mmol/L Intact Control  30 ± 3** 15.4 ±1.3 0.09 ± 0.03** 33.7 ± 0.7** 2.28 ± 0.12 1.4 ± 0.2 OVX Control 51 ± 411.7 ± 1.2 3.55 ± 0.50  44.0 ± 0.9  2.29 ± 0.16 1.1 ± 0.1 OVX + MPA 57 ±4 11.7 ± 1.2 2.51 ± 0.20*  35.5 ± 0.7** 2.55 ± 0.14 1.3 ± 0.1 OVX + E₂41 ± 5  9.2 ± 0.9 2.37 ± 0.45*  40.4 ± 0.8** 2.02 ± 0.15 0.8 ± 0.1 OVX +DHT 56 ± 5  7.8 ± 0.7* 1.55 ± 0.27** 38.8 ± 0.7** 2.44 ± 0.16 0.9 ± 0.1OVX + DHEA  201 ± 25**  7.3 ± 1.0* 0.02 ± 0.01** 34.5 ± 0.7**  1.78 ±0.16* 0.8 ± 0.1 OVX + DHEA + FLU  103 ± 10** 14.5 ± 1.2 1.13 ± 0.24**41.5 ± 0.7*  2.27 ± 0.15 0.8 ± 0.1 OVX + DHEA + EM-800  202 ± 17**   6.4± 1.0** LD** 39.2 ± 0.7**  0.63 ± 0.09** 1.0 ± 0.2 LD: Limit ofDetection: 0.01 ng/ml *p < 0.05; **p < 0.01 versus OVX Control

Example 6 Example of Synthesis of the Preferred Compound of theInvention Synthesis of(S)-(+)-7-hydroxy-3-(4′-hydroxyphenyl)-4-methyl-2-(4″-(2′″-piperidinoethoxy)phenyl)-2H-1-benzopyranhydrochloride EM-01538 (EM-652, HCl)

Step A: BF₃.Et₂O, toluene; 100° C.; 1 hour.

Step C: 3,4-dihydropyran, p-toluenesulfonic acid monohydrate, ethylacetate; 25° C. under nitrogen, 16 hours, and then crystallization inisopropanol.

Steps D, E, and F:

(1) piperidine, toluene, Dean & Stark apparatus, reflux under nitrogen;(2) 1,8-diazabicyclo[5,4,0]undec-7-ene, DMF, reflux 3 hours;

(3) CH₃MgCl, THF, −20 to 0° C. and then room temperature for 24 hours;

Steps G, H: (1S)-(+)-10-camphorsulfonic acid, acetone, water, toluene,room temperature, 48 hours.

Step HH: 95% ethanol, 70° C., then room temperature 3 days.

Step HHR: Recycling of mother liquor and wash of step HH

(S)-10-camphorsulfonic acid, reflux; 36 hours, then room temperature for16 hours.

Step I:

(1) DMF aq., Na₂CO₃, ethyl acetate;

(2) ethanol, dilute HCl;

(3) water.

Synthesis of2-tetrahydropyranyloxy-4-hydroxy-2′-(4″-tetrahydropyranyloxyphenyl)acetophenone (4). A suspension of2,4-dihydroxy-2′-(4″-hydroxyphenyl)acetophenone 3 (97.6 g, 0.4 mole)(available from Chemsyn Science Laboratories, Lenexa, Kans.) in3,4-dihydropyran (218 ml, 3.39 mole) and ethyl acetate (520 ml) wastreated with p-toluenesulfonic acid monohydrate (0.03 g, 0.158 mmole) atabout 25° C. The reaction mixture was stirred under nitrogen with noexternal heating for about 16 hours. The mixture was then washed with asolution of sodium bicarbonate (1 g) and sodium chloride (5 g) in water(100 ml). The phases were separated and the organic phase was washedwith brine (20 ml). Each wash was back extracted with 50 ml ethylacetate. All the organic phases were combined and filtered throughsodium sulfate.

Solvent (about 600 ml) was removed by distillation at atmosphericpressure and isopropanol (250 ml) was added. Additional solvent (about300 ml) was distilled at atmospheric pressure and isopropanol (250 ml)was added. Additional solvent (about 275 ml) was distilled atatmospheric pressure and isopropanol (250 ml) was added. The solutionwas cooled at about 25° C. with stirring and after about 12 hours, thecrystalline solid was filtered, washed with isopropanol and dried (116.5g, 70%).

Synthesis of4-hydroxy-4-methyl-2-(4′-[2″-piperidino]-ethoxy)phenyl-3-(4′″-tetrahydropyranyloxy)phenyl-7-tetrahydropyranyloxy-chromane(10). A solution of2-tetrahydropyranyloxy-4-hydroxy-2′-(4″-tetrahydropyranyloxyphenyl)acetophenone4 (1 kg, 2.42 mole), 4-[2-(1-piperidino)ethoxy]benzaldehyde 5 (594 g,2.55 mole) (available from Chemsyn Science Laboratories, Lenexa, Kans.)and piperidine (82.4 g, 0.97 mole) (available from Aldrich ChemicalCompany Inc., Milwaukee, Wis.) in toluene (8 L) was refluxed undernitrogen with a Dean & Stark apparatus until one equivalent of water (44mL) was collected.

Toluene (6.5 L) was removed from the solution by distillation atatmospheric pressure. Dimethylformamide (6.5 L) and1,8-diazabicyclo[5,4,0]undec-7-ene (110.5 g, 0.726 mole) were added. Thesolution was agitated for about 8 hours at room temperature to isomerizethe chalcone 8 to chromanone 9 and then added to a mixture of water andice (8 L) and toluene (4 L). The phases were separated and the toluenelayer washed with water (5 L). The combined aqueous washes wereextracted with toluene (3×4 L). The combined toluene extracts werefinally washed with brine (3×4 L), concentrated at atmospheric pressureto 5.5 L and then cooled to −10° C.

With continued external cooling and stirring under nitrogen, a 3Msolution of methylmagnesium chloride in THF (2.5 L, 7.5 mole) (availablefrom Aldrich Chemical Company Inc., Milwaukee, Wis.) was added,maintaining the temperature below 0° C. After all the Grignard reagentwas added, the external cooling was removed and the mixture allowed warmto room temperature. The mixture was stirred at this temperature forabout 24 hours.

The mixture was again cooled to about −20° C. and with continuedexternal cooling and stirring, saturated ammonium chloride solution (200ml) was added slowly, maintaining the temperature below 20° C. Themixture was stirred for 2 hours and then added the saturated ammoniumchloride solution (2 L) and toluene (4 L) and agitated for five minutes.The phases were separated and the aqueous layer extracted with toluene(2×4 L). The combined toluene extracts were washed with dilutehydrochloric acid until the solution became homogenous and then withbrine (3×4 L). The toluene solution was finally concentrated atatmospheric pressure to 2 L. This solution was used directly in the nextstep.

Synthesis of(2R,S)-7-hydroxy-3-(4′-hydroxyphenyl)-4-methyl-2-(4″-[2′″-piperidino]ethoxy)phenyl)-2H-1-benzopyran(1S)-10-camphorsulphonic acid salt (±12). To the toluene solution of4-hydroxy-4-methyl-2-(4′-[-2″-piperidino]-ethoxy)-phenyl-3-(4′″-tetrahydropyranyloxy)phenyl-7-tetrahydropyranyloxychromane(10) was added acetone (6 L), water (0.3 L) and (S)-10-camphorsulphonicacid (561 g, 2.42 mole) (available from Aldrich Chemical Company Inc.,Milwaukee, Wis.). The mixture was agitated under nitrogen for 48 hoursafter which time the solid(2R,S)-7-hydroxy-3-(4′-hydroxyphenyl)-4-methyl-2-(4″-[2′″-piperidino]ethoxy)phenyl)-2H-1-benzopyran(1S)-10-camphorsulphonic acid salt (12) was filtered, washed withacetone and dried (883 g). This material was used in the next (HH) stepwithout further purification.

Synthesis of(2S)-7-hydroxy-3-(4′-hydroxyphenyl)-4-methyl-2-(4″-[2′″-piperidino]ethoxy)phenyl)-2H-1-benzopyran(1S)-10-camphorsulphonic acid salt (13, (+)-EM-652(1S)-CSA salt). Asuspension of(2R,S)-7-hydroxy-3-(4′-hydroxyphenyl)-4-methyl-2-(4″-[2′″-piperidino]ethoxy)phenyl)-2H-benzopyran(1S)-10-camphorsulphonic acid salt±12 (759 g) in 95% ethanol was heatedwith stirring to about 70° C. until the solid had dissolved. Thesolution was allowed to cool to room temperature with stirring thenseeded with a few crystals of(2S)-7-hydroxy-3-(4′-hydroxyphenyl)-4-methyl-2-(4″-[2′″-piperidino]ethoxy)phenyl)-2H-1-benzopyran(1S)-10-camphorsulphonic acid salt 13. The solution was stirred at roomtemperature for about three days in total. The crystals were filtered,washed with 95% ethanol and dried (291 g, 76%). The de of the productwas 94.2% and the purity 98.8%.

Synthesis of(S)-(+)-7-hydroxy-3-(4′-hydroxyphenyl)-4-methyl-2-(4″-(2′″-piperidinoethoxy)phenyl)-2H-1-benzopyranhydrochloride EM-01538 (EM-652, HCl). A suspension of compound 13(EM-652-(+)-CSA salt, 500 mg, 0.726 mmol) in dimethylformamide (11 μL,0.15 mmol) was treated with an 0.5 M aqueous sodium carbonate solution(7.0 mL, 3.6 mmol), and stirred for 15 min. The suspension was treatedwith ethyl acetate (7.0 mL) and stirred during 4 h. The organic phasewas then washed with an aqueous saturated sodium carbonate solution (2×5mL) and brine (1×5 mL) dried over magnesium sulfate, and concentrated. Asolution of the resulting pink foam (EM-652) in ethanol (2 mL) wastreated with 2 N hydrochloric acid (400 μL, 0.80 mmol), stirred for 1 h,treated with distilled water (5 mL), and stirred during 30 min. Theresulting suspension was filtered, washed with distilled water (5 mL),dried in air and under high vacuum (65° C.) to give a creamy powder (276mg, 77%): Fine off-white powder; Scanning Calorimetry: Melting peakonset at 219° C., ΔH=83 J/g; [α]²⁴ _(D)=154° in methanol 10 mg/ml.; ¹HNMR (300 MHz, CD₃OD) δ (ppm) 1.6 (broad, 2H, H-4′″), 1.85 (broad, 4H,H-3″″ and 5″″), 2.03 (s, 3H, CH₃), 3.0 and 3.45 (broad, 4H, H-2″″ and6″″), 3.47 (t, J=4.9 Hz, 2H, H-3′″), 4.26 (t, J=4.9 Hz, 2H, H-2′″), 5.82(s, 1H, H-2), 6.10 (d, J=2.3 Hz, 1H, H-8), 6.35 (dd, J=8.4, 2.43 Hz, 1H,H-6), 6.70 (d, J=8.6 Hz, 2H, H-3′, and H-5′), 6.83 (d, J=8.7 Hz, 2H,H-3″ and H-5″), 7.01 (d, J=8.5 Hz, 2H, H-2′ and H-6′), 7.12 (d, J=8.4Hz, 1H, H-5), 7.24 (d, J=8.6 Hz, 2H, H-2″ and H-6″); ¹³C RMN (CD₃OD, 75MHz) δ ppm 14.84, 22.50, 23.99, 54.78, 57.03, 62.97, 81.22, 104.38,109.11, 115.35, 116.01, 118.68, 125.78, 126.33, 130.26, 130.72, 131.29,131.59, 134.26, 154.42, 157.56, 158.96, 159.33. Elemental Composition:C, H, N, Cl: Theory; 70.51, 6.53, 2.84, 7.18, %. Found: 70.31, 6.75,2.65, 6.89%.

Example 7 In Vivo Assays of Bioavailability of the Prodrugs ofAndrost-5-ene-3β,17β-diol

1) Principle

The assays of the bioavailability of prodrugs of sex steroid precursorswere performed in male Sprague Dawley rats by measuring the plasmaconcentrations of the compounds after single oral administration of thecompounds.

a) Animals and Treatment

Male Sprague-Dawley rats [Crl:CD(SD)Br] weighing 275-350 g were obtainedfrom Charles-River Canada Inc. and housed 2 per cage during theacclimation period and individually during the study period. The animalswere maintained under a regimen of 12 hours light: 12 hours dark (lightson at 08:00). Animals received certified Rodent feed (Lab Diet #5002,pellets) and tap water ad libitum. Rats were fasted (access to wateronly) starting on the evening prior to dosing.

Each compound to be tested was administered to three animals as asuspension in 0.4% methylcellulose by oral gavage at a dose of 150μmg/rat. One blood sample of ˜0.7 ml was collected from the jugular veinof rats under Isoflurane-induced anesthesia at 1, 2, 3, 4, and 7 hourspost-gavage. Blood samples were immediately transferred into arefrigerated 0.75 ml Microtainer containing EDTA and kept in anice-water bath until centrifugation at 3000 rpm for 10 minutes. Plasmaseparation was performed rapidly (less than 50 minutes) after bloodcollection. One aliquot of 0.25 ml of plasma was then transferred into aborosilicate tube (13×100) and was rapidly frozen on dry-ice. Plasmasamples were kept at −80° C. until measurement of plasma concentrationof the sex steroid or sex steroid precursors by GC-MS.

Results:

Oral absorption and AUCs are shown in FIGS. 12 and 13.

Pharmaceutical Composition Examples

Set forth below, by way of example and not of limitation, are severalpharmaceutical compositions utilizing preferred active SERM EM-800 orEM-1538 and preferred active a sex steroid precursor DHEA, EM-1304 orEM-01474-D Other compounds of the invention or combination thereof, maybe used in place of (or in addition to) EM-800 or EM-1538, DHEA, EM-1304or EM-01474-D. The concentration of active ingredient may be varied overa wide range as discussed herein. The amounts and types of otheringredients that may be included are well known in the art.

Example A

Tablet Weight % Ingredient (by weight of total composition) EM-800 5.0DHEA 15.0 Gelatin 5.0 Lactose 58.5 Starch 16.5

Example B

Gelatin capsule Weight % Ingredient (by weight of total composition)EM-800 5.0 DHEA 15.0 Lactose hydrous 65.0 Starch 4.8 Cellulosemicrocrystalline 9.8 Magnesium stearate 0.4

Kit Examples

Set forth below, by way of example and not of limitation, are severalkits utilizing preferred active SERM EM-800 or EM-1538 and preferredactive a sex steroid precursor DHEA, EM-1304 or EM-01474-D Othercompounds of the invention or combination thereof, may be used in placeof (or in addition to) EM-800 or EM-1538, DHEA, EM-1304 or EM-01474-D.The concentration of active ingredient may be varied over a wide rangeas discussed herein. The amounts and types of other ingredients that maybe included are well known in the art.

Example A

The SERM is orally administered while the sex steroid precursor ispercutaneously administered

SERM composition for oral administration (capsules) Weight % Ingredient(by weight of total composition) EM-800 5.0 Lactose hydrous 80.0 Starch4.8 Cellulose microcrystalline 9.8 Magnesium stearate 0.4

Sex steroid precursor composition for topical administration (gel)Weight % Ingredient (by weight of total composition) DHEA 10.0Caprylic-capric Triglyceride 5.0 (Neobee M-5) Hexylene Glycol 15.0Transcutol (diethyleneglycol 5.0 monomethyl ether) Benzyl alcohol 2.0Cyclomethicone (Dow corning 345) 5.0 Ethanol (absolute) 56.0Hydroxypropylcellulose (1500 cps) 2.0 (KLUCEL)

Example B

The SERM and the sex steroid precursor are orally administeredNon-Steroidal Antiestrogen composition for oral administration(capsules) Weight % Ingredient (by weight of total composition) EM-8005.0 Lactose hydrous 80.0 Starch 4.8 Cellulose microcrystalline 9.8Magnesium stearate 0.4

Sex steroid precursor composition for oral administration (Gelatincapsule) Weight % Ingredient (by weight of total composition) DHEA 15.0Lactose hydrous 70.0 Starch 4.8 Cellulose microcrystalline 9.8 Magnesiumstearate 0.4

Other SERMs may be substituted for EM-800 or EM-01538 in the aboveformulations, as well as other sex steroid inhibitors may be substitutedfor DHEA, EM-1304 or EM-01474-D. More than one SERM or more than oneprecursor may be included in which case the combined weight percentageis preferably that of the weight percentage for the single precursor orsingle SERM given in the examples above.

The invention has been described in terms of preferred embodiments andexamples, but is not limited thereby. Those of skill in the art willreadily recognize the broader applicability and scope of the inventionwhich is limited only by the patent claims herein.

1. A method of treating ovarian cancer, said method comprisingadministering to a patient in need of said treatmentdehydroepiandrosterone, and further comprising administering to saidpatient a therapeutically effective amount of a selective estrogenreceptor modulator that is a pharmaceutically acceptable salt of:

as part of a combination therapy.
 2. The method of claim 1 wherein theselective estrogen receptor modulator is: